Keywords

1 Mucosal Immune System

The first productive interaction between most infectious agents and the host is with mucosal surfaces, specially, the nasal, oropharyngeal, respiratory, genitourinary, and gastrointestinal mucosa. Conventional vaccine strategies that involve parenteral immunization with inactivated viruses or bacteria or subunits of relevant virulence determinants of those pathogens do not prevent initial interactions. In-fact, traditional vaccine strategies do not prevent infection but instead resolve infection before disease ensues. Moreover, many bacterial toxins bind to and interact with mucosal epithelial cells, in which case significant damage to the host may ensue before serum antibodies can play a role in protection. The mucosal surfaces of the gastrointestinal and respiratory tracts represent the main portals of entry for most human pathogens. Sexual contact is another mucosal mode of transmission of infection. Direct inoculation of pathogens into the bloodstream is other important route of infection. Most external mucosal surfaces are replete with organized follicles and scattered antigen-reactive or sensitized lymphoid elements, including B cells, T lymphocytes, T-cell subsets, plasma cells, and a variety of other cellular elements involved in the induction and maintenance of immune response. The mucosal surfaces encompass a critical component of the mammalian immunologic repertoire.

Numerous studies have indicated that induction of systemic immunity through parenteral immunization can effectively clear systemic infections, but it usually fails to protect the mucosal surfaces. Mucosal vaccine administration with an appropriate adjuvant, on the other hand, can induce immune responses at both systemic and mucosal sites and as a result, may prevent not only infectious diseases but also colonization at mucosal surfaces [1]. The mucosal immune system differs in several ways from the systemic immune system. Mucosal immunization frequently results in the stimulation of both mucosal and systemic immune responses, while systemic immunization typically only induces systemic responses without activating the mucosal immune system. Induction of mucosal response leads to production of secretory IgA (sIgA) antibodies, which are not usually produced by systemic immunization [2]. The production of sIgA on the mucosal surfaces is result of the local exposure of antigens to the mucosal-associated lymphoid tissues, especially those in the upper respiratory tract, and the gastrointestinal tract. In most cases infectious agents enter the body at mucosal surfaces and therefore the protective immunity at these surfaces can be effectively induced by mucosal immunization through oral, nasal, rectal, or vaginal routes [3].

The stimulation of the mucosal immune system at one mucosal site can lead to sIgA production in the local as well as distal mucosal surfaces. For example, antigen stimulation of the Peyer’s patches in the gastrointestinal tract produced sIgA-producing B cells not only in the intestine, but also in the bronchi as well as in the genitourinary tract. This interconnected mucosal system of sIgA induction and production is termed as common mucosal immune system [2, 4]. The immunologic network operating on external mucosal surfaces consists of gut-associated lymphoid tissue (GALT), the lymphoid structures associated with bronchoepithelium and lower respiratory tract (BALT), ocular tissue, upper airway, salivary glands, tonsils, and nasopharynx (nasal associated lymphoid tissue; NALT), larynx (LALT), middle ear cavity, male and female genital tracts, and mammary glands. The organized lymphoid follicles in the GALT and BALT are main inductive sites of mucosal immune response.

2 Mucosal Immunization: An Edge Over Parenteral Vaccination

Vaccine delivery via mucosal route has several advantages over parenteral vaccination [5]. The most important reason for using a mucosal route of vaccination instead of a parenteral route is that the vast majority of infections occurs at or takes their departure from mucosal surfaces and in these infections mucosal vaccines are usually required to induce a protective immune response. The parenteral immunization induces poor mucosal immunity; however, mucosal immunization can induce both mucosal and systemic immunity [6]. The immunization at one mucosal site can induce specific responses at distant sites because of the expression of mucosa-specific homing receptors (site-specific integrins) by mucosally primed lymphocytes and complementary mucosal-tissue specific receptors (addressins) on the vascular endothelial cells [7]. This interconnected network is important because protective immunity (for instance against sexually transmitted diseases) could be induced in segregated mucosal sites in a practical way such as by oral or intranasal immunization and without hampering cultural or religious barriers. Mucosal vaccines are potentially useful to overcome the known barrier of parenteral vaccination caused by either preexisting systemic immunity from previous vaccination or in young children from maternal antibodies or selective immunosuppression such as that caused by HIV infection. For example, mucosal antibody response to oral cholera vaccination was observed in AIDS patients even after they had completely lost their ability to respond to an injectable vaccine (tetanus toxoid) [8]. In addition to serum IgG and mucosal IgA antibodies, mucosal immunization can stimulate cell mediated responses including helper CD4+ T cells and CD8+ cytotoxic T lymphocytes, the latter being important to eliminate intracellular pathogens [9]. The mucosal vaccine delivery is crucial for protective efficacy against noninvasive infections at mucosal surfaces that are normally impermeable to serum antibodies transduction, or passive passage across an epithelium, e.g., GIT infection with V. cholerae [7]. The mucosal vaccine delivery is particularly important for pathogens that can infect the host through both systemic and mucosal route because induction of both sIgA and systemic IgG confer protection at both site. This mode of vaccine delivery could be explored for combating pathogens acquired through non-mucosal routes such as blood or skin. Mucosal vaccination is also beneficial to induce peripheral systemic tolerance especially against those T cell mediated immune reaction that are associated with development of delayed type hypersensitivity reactions. This strategy is important to avoid delayed type hypersensitivity reactions and other allergic reactions to many ingested food proteins and other allergens [6]. Mucosal tolerance is a specific systemic hyporesponsiveness that arises after mucosal administration of an antigen. The tolerance is mediated by a combination of suppressor T-cells, inhibitory cytokines and factors which inhibit the inflammatory process. Oral tolerance can be used for the treatment of atopic diseases in human [10]. Oral tolerance varies when the antigen is administered in a soluble form as compared to a particulate state.

However, one major limitation with mucosal immunization is a striking balance between achieving an effective therapeutic response with a particular dose and preventing the induction of tolerance. Therefore, it is important to understand the mechanisms involved in controlling these responses. The key cells that determine the success or demise of a vaccine are antigen-presenting cells (APC), e.g., dendritic cells and T lymphocytes, including sub-populations of T-helper cells, T-cytotoxic cells, and regulatory T cells [7]. Immune interactions at the local level will have a profound effects on the type of immune response generated (e.g., first nuclear factor of activated T cell proteins is involved in both the generation of Th1 or Th2 cells and the maintenance of T-cell tolerance), so a clear understanding of local immune responses at the site of antigen uptake is essential [11].

On the other hand mucosal administration of vaccines also offers a number of practical advantages. Mucosal vaccination, being noninvasive in nature, does not require the use of needles. This would carry less risk of transmitting type of infections still associated with needle reuse [12, 13]. Additionally, mucosal vaccination is relatively easy and does not require expensive trained personnel. The production of mucosal vaccines may be cost effective in comparison to injectable vaccines that require high standards of purity, in addition to sterility. Moreover oral vaccines can also be expected to have much greater acceptability than injectable vaccines by causing no sore arm etc. Further it can enhance vaccine safety and adverse effect by avoiding direct contact between potentially toxic vaccine component and systemic circulation. Finally, mucosal vaccines allow for the easy administration of multiple vaccines [14].

3 M Cell as Gateway of Mucosal Immune Systems

The common features of all inductive mucosal sites include epithelial surface containing M cells overlying organized lymphoid follicles (Fig. 9.1). M cells offer functional openings in the epithelial barrier through vesicular transport activity [15]. They are well suited for efficient endocytosis and transcytosis. The M cells lack the rigid brush border cytoskeleton of their enterocyte neighbors, and their apical surfaces have broad membrane, a microdomain from which endocytosis occurs. The M cell basolateral membrane is deeply invaginated to form a large intraepithelial “pocket” containing T lymphocytes, B lymphocytes, and macrophages. This structural specialization brings the basolateral cell surface to within a few microns of the apical surface and greatly shortens the distance that transcytotic vesicles must travel to cross the epithelial barrier. Endocytic or phagocytic uptake of foreign antigens or particles is followed by rapid transcytosis directly to the intraepithelial pocket, with little or no retention in M cell lysosomes. After M cell transport, antigens are processed and presented by macrophages, dendritic cells, and B cells within and below the epithelium, resulting in generation of IgA-committed, antigen-specific B lymphoblast that proliferate locally in the germinal centers of O-MALT and migrate via the bloodstream to distant mucosal and glandular tissues, where they differentiate into plasma cells. The dimeric or polymeric IgA antibodies thus produced are selectively bound by epithelial polymeric immunoglobulin receptors, transcytosed across epithelial cells, and released into glandular and mucosal secretions [16]. Thus, M cell plays a pivotal role in the elicitation of secretory immune response.

Fig. 9.1
figure 1

M cells of the Peyer’s patches

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4 Distinguishing Feature of M Cells

The M cell apical surfaces are distinguished from their enterocyte neighbors by the absence of a typical brush border and the presence of variable microvilli [17] and a unique intraepithelial “pocket.” This hallmark structural feature of fully differentiated M cells, provides a docking site for lymphocytes and shortens the distance that transcytotic vesicles must travel from the apical to the basolateral surface. The basolateral surface of the M cell includes the two major subdomains typical of all epithelial cells: the lateral subdomain is involved in cell–cell adhesion and contains Na + -K + -ATPase, and the basal subdomain interacts with the extracellular matrix and basal lamina.

The M cell apical surface differs from that of intestinal absorptive cells in other respects as well. Most M cells in Peyer’s patches lack the highly organized brush border with uniform, closely packed microvilli typical of enterocytes. The actin-associated protein villin, confined to microvilli in enterocytes, is diffusely distributed in M cells [18], reflecting the modified apical organization and perhaps the ability to rapidly respond to adherence of microorganisms with ruffling and phagocytosis. M cells are also recognized by their lack of certain enterocytes surface glycoproteins. Whereas enterocyte brush borders have abundant hydrolytic enzymes, these enzymes are usually reduced or absent on M cells [19, 20]. In addition, the thick filamentous glycocalyx typical of enterocytes is often absent from M cells, rendering the M cell surface more accessible to luminal materials. Further, M cell apical surfaces are coated with glycoproteins that display glycosylation patterns different from their enterocyte neighbors, and although the protein backbones have not been identified, the carbohydrate epitopes can be useful M cell identifiers which can be explored for targeted vaccine delivery.

5 M-Cell Surface Receptors

On the basis of the observation that M cells are targeted by a variety of soluble tracers and inert particles, it has been suggested that M cell targeting by microorganisms may be mediated, at least in part, by nonspecific, passive mechanisms. Such mechanisms are likely to be influenced by the physical properties of the pathogen, since the interaction of inert particles with M cells is influenced by the physicochemical properties of the particle preparation and by species related variations in M cell surface properties [21–24]. Additionally, specific receptors located in either the M cell apical membranes or the closely associated glycocalyx also contribute to M cell targeting by microorganisms. In the view of the fact that M cells bind and endocytose antibodies located in the lumen, there is interest in ascertaining whether there are specific Ig receptors on M cells and the follicle associated epithelium (FAE). A novel IgA receptor was discovered in mouse M cells with the potential to facilitate transport of sIgA from luminal secretions into GALT [25]. The finding that sialylated Lewis A antigen (SLAA) appeared to be expressed selectively on M cells from a small number of biopsies of human Peyer’s and cecal patches was very exciting [26]. Although a “universal” M-cell marker is lacking, some exciting results from targeting experiments have been observed. For example, the coupling of recombinant cholera toxin B subunit to liposomes containing Streptococcus mutans antigens generated enhanced mucosal immunization in mice compared to untargeted antigen-loaded particles [27].

Ulex europaeus agglutinin 1 (UEA-1) has high specificity for the carbohydrate moiety, α-l-fucose, located on the apical membranes of mouse M cells [28, 29]. There have been successful efforts made into in vivo targeting to mouse M cells by conjugating the lectin to polymerized liposomes [30] and also to latex particles [31]. There are many success stories of UEA-1based delivery system is animal models; however, it is of limited value in vaccine delivery in human because the lectin is toxic, is subject to intestinal degradation, and its receptor is not expressed in human PP [26]. As another alternative to the lectin UEA-1, the edible orange peel mushroom Aleuria aurantia was used to target the α-l-fucose receptor. Coated poly(d,l-lactide-co-glycolide) (PLGA) particles were entrapped with birch pollen antigens and administered to mice as a potential oral allergen immunotherapy [32]. M cells can also be discriminated from enterocytes in the FAE on the basis of altered adherens junction protein expression [33]. Various other receptors and markers on FAE or M cells from various species have been summarized by Brayden et al. 2005 [34].

6 Mucosal Vaccine Delivery Systems

The mucosal vaccine delivery systems can be classified into live vectors (e.g., Salmonella typhi) and nonliving antigen delivery systems (Liposomes, nanoparticles, immunostimulating complexes, etc.). These mucosal vaccine carriers are summarized in Table 9.1, and in this chapter two potential mucosal vaccine delivery systems, i.e., polymeric particles and liposomes are described.

Table 9.1 Various options for the vaccine delivery by mucosal route
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6.1 Polymeric Particles

It has been demonstrated that the incorporation of an antigen into microparticles [49], or its adsorption to the surface of biodegradable microparticles [50], resulted in the induction of enhanced serum and secretory antibody responses, following oral administration. Subsequently, it was shown that oral delivery of an antigen entrapped in PLGA microparticles also resulted in the induction of enhanced immunity [51]. During last two decades biodegradable PLGA based microparticles/nanoparticles have been explored extensively [52–54]. Binding and uptake of the particles was enhanced when particles were conjugated to B subunit of E. coli heat labile enterotoxin (LTB), the plant lectin, ConA or vitamin B12 following oral delivery to rats [55]. Covalent attachment of UEA-1 to polystyrene microspheres and oral delivery to mice result in selective binding to and rapid uptake by the Peyer’s patch M-cells [56]. Orally administered polystyrene microparticles with attached Lycopersicon esculentum agglutinin (LEA) were taken to a greater extent than unconjugated particles in the rats [57]. The linkage of sepharose beads to WGA and Solanum tuberosum lectin (STL) enhanced their binding to caco-2 cells [58]. These observations and a body of additional data suggest that lectins are potential tools for the enhanced binding and internalization of orally delivered drugs and drug delivery systems and efficiency of oral vaccines can be improved by M-cell targeting.

We have described the development of PLGA nanoparticles loaded with HBsAg and the antigen stabilization in the presence of trehalose and Mg(OH)2. Additionally, UEA-1 lectin was anchored to the nanoparticles to target them to M-cells of the Peyer’s patches [59]. The results suggest that HBsAg can be successfully stabilized by co-encapsulation of protein stabilizers. The lectinized nanoparticles have demonstrated approximately fourfold increase in the degree of interaction with the bovine submaxillary mucin (BSM) as compared to plain nanoparticles and sugar specificity of the lectinized nanoparticles was also maintained. The serum anti-HBsAg titre obtained after oral immunization with Hepatitis B surface antigen (HBsAg) loaded stabilized lectinized nanoparticles was comparable with the titre recorded after alum-HBsAg given intramuscularly. The stabilized UEA-1 coupled nanoparticles exhibited enhanced immune response as compared to stabilized non-lectinized nanoparticles. Furthermore, the stabilized lectinized nanoparticles elicited sIgA in the mucosal secretion and IL-2 and IFN-γ in the spleen homogenates.

6.1.1 Factors Affecting Uptake of Polymeric Particles

The M cell appears to be the primary route of entry into the host for several enteric viral pathogens. The mechanism for the uptake of synthetic and biodegradable microparticles by M cells appears similar to that observed for bacteria. Microparticle uptake initially involves contact with the microvillus projections on the M cell surface followed by rapid phagocytosis through the extension of apical membrane processes. Various factors affecting the uptake of particles are described below.

6.1.1.1 Particle Size

In general, smaller microparticles are absorbed to a greater degree than larger microparticles. The smaller particles are distributed more easily to distant sites, and remain detectable for longer periods of time. These conclusions are consistent for microparticles made of different polymers and different size ranges. Jani et al. [60] studied the comparative uptake of 50 nm, 500 nm, and 1 μm polystyrene particles and found that 50 nm particles are absorbed and distributed quicker than 500 nm and 1 μm particles. Florence suggested that decrease in particle diameter may result in increased uptake below 1 μm and particles above 3 μm are taken up by the Peyer’s patches but remained there [61].

6.1.1.2 Hydrophobicity

Hydrophobicity of the particles influences profoundly their uptake behavior. Jung et al. reported that uptake of nanoparticles prepared from hydrophobic polymer was higher than from particles with more hydrophilic surfaces [62]. They further added that hydrophobic polystyrene nanoparticles interact with M cells with more affinity than absorptive epithelia whereas less hydrophobic PLGA nanoparticles interact with both cell types. Other investigators have shown that decreasing surface hydrophobicity, by the adsorption of poloxamers 235, 238, 407, or poloxamines 901, 904, and 908, may decrease the uptake of polystyrene microparticles into cells of the immune system, thereby avoiding elimination [63]. The charge on the particles also determines their uptake by the intestinal epithelia. Although the charged particles are taken up, their uptake was less than the non-ionic hydrophobic particles [61]. The negatively charged and neutral particles exhibited greater affinity to PP in comparison to positively charged particles [64]. This finding was in accordance with previous report that a combination of both, negative charge and increased hydrophobicity of the particles improve the gastrointestinal uptake [62].

6.1.1.3 Effect of Dose and Vehicle on Uptake

The extent of particle uptake is also influence by the dosing. It was observed that polystyrene particles were identified in Peyer’s patches with difficulty after 1 day of feeding, but were readily identified following chronic feeding. Le Ray et al. [65] have shown that changing the vehicle in which the particles were administered could enhance the extent of uptake of polystyrene particles in mice. Further, volume and tonicity of the administered vehicle also have an effect on the extent of uptake [66].

6.1.1.4 Glass Transition Temperature and Crystallinity of the Polymer

Glass transition temperature (Tg) and crystallinity of the polymer are two important bulk properties of the polymer affecting the release of incorporated components. Without proper release characteristics, drugs or vaccines incorporated into microparticles may be released either prematurely or to insignificant levels before elimination. Tg is the temperature at which a transition occurs from the glassy state to the rubbery state resulting in increase in the molecular motion and free volume of the amorphous polymer, which in turn increases the drug release from the polymer. Above Tg the polymer acquires sufficient thermal energy for isomeric rotational motion or for significant torsional oscillation to occur about most of the bonds in the main chain. This leads to an increase in the free volume of the amorphous polymer, and thus in turn, the release of incorporated bioactive [67].

Migliaresi et al. observed an increase in the degree of crystallinity of polylactic acid with the degradation of the polymer [68]. This could be related to the faster degradation of the amorphous phase of the semicrystalline polymer, resulting in loss of amorphous material and a concomitant increase in crystallinity. A decrease in crystallinity increases the drug release because the diffusion coefficient and solubility of the drug in polymer are inversely proportional to at least the first power of the amorphous content. The structural features, which influence the crystallinity of the polymer, are similar to those, which affect the glass transition temperature.

Bioactive (drug/vaccine) may release from nanoparticles/microparticles by several mechanisms including surface and bulk erosion, disintegration, microparticle hydration, drug diffusion and desorption. These bioactive release mechanisms are in turn controlled by bulk properties such as the molecular weight of the polymer (affecting crystallinity and glass transition temperature), the copolymer composition, polymer matrix density and the extent and nature of the cross-linking. By adjusting the blend ratio of PLGA/polyethyleneglycol (PEG) the release profile of entrapped dextran and rabbit gamma immunoglobulin (IgG) microparticles can be varied [69]. The release rate of entrapped compounds increased with increasing PEG content because of the leaching out of PEG from the polymer blend into the aqueous phase during drug release, resulting in the increased porosity of microparticles. Changing the monomer ratio of lactide/glycolide in PLGA microparticles from 75:25 to 50:50 led to an increase in the release rate of entrapped OVA due to an increase in the degradation rate of the microparticles [70].

6.1.1.5 Effect of Additives

Various additives are involved in the fabrication of nanoparticles/microparticles. Polyvinyl alcohol (PVA) is the most commonly used emulsifier in the formulation of lactide and poly (d,l-lactide-co-glycolide) nanoparticles/microparticles. A fraction of PVA remains associated with the nanoparticles/microparticles despite repeated washing because PVA forms an interconnected network with the polymer at the interface. The residual PVA affect different pharmaceutical properties of the particles such as particle size, zeta potential, polydispersity index, surface hydrophobicity, protein loading and also slightly influenced the in-vitro release of encapsulated protein. Importantly, nanoparticles with higher amount of residual PVA had relatively lower cellular uptake despite their smaller particle size [71]. The lower cellular uptake of nanoparticles with higher amount of residual PVA is attributed to the higher hydrophilicity of the nanoparticle surface. Trehalose is well-documented protein stabilizer. We have observed increase in the release of HBsAg with trehalose stabilized PLGA nanoparticles/microparticles when compared with PLGA nanoparticles/microparticles without trehalose [72]. Since the protein stabilizer (trehalose) reduced denaturation at the aqueous-organic interface, the payload of HBsAg was increased and this was reflected in augmented cumulative percent release. Moreover, sugars (e.g., trehalose, sucrose) have appreciable solubility in aqueous media. They dissolve rapidly from the matrix leaving a porous matrix, which in turn releases antigen/bioactive relatively faster.

6.1.1.6 Effect of Species, Animal Age, and Food Ingestion on Uptake

The species variation can affect uptake extent of the particles. The uptake of polystyrene particles in rabbit was at least an order of magnitude greater than mice because of the greater abundance of the M cells in the Peyer’s patches [73]. Le Fevre et al. showed greater uptake of polystyrene particles in older mice [74]. Other investigator reported that age of the animal did not affect the extent of polystyrene particle uptake in rats [75]. The extent of uptake in the mice was enhanced by the presence of food, which may delay the intestinal transit of the particles [75].

6.1.1.7 Intestinal Mucus Layer Characteristics

The uptake of the nanoparticles/microparticles is preceded by their passage through two barriers, i.e., the mucus gel layer and the mucosa. Intestinal mucus is a high molecular weight glycoprotein secretion, which covers the mucosa with a continuous adherent blanket. The mucus layer protects the gastrointestinal mucosa from potentially harmful bacteria, pathogens, or chemicals [76]. Several investigators have reported diminished diffusion of small and large compounds such as bovine serum albumin (BSA), lysozyme, tertiary amines, and quaternary ammonium compounds [77]. Mucus acts as a barrier by entrapping microparticles, causing agglomeration, which results in an increase in net size and a resultant decrease in diffusion coefficient, and by decreasing the diffusion coefficient through the mucus thereby restricting diffusion to the mucosa layer. Since the high number of sulfate, sialic acid, and sugar moieties in the carbohydrate side chains of the mucin molecule impart a highly negative charge to mucin [76], it may be expected that electrostatic interactions between positively charged drugs and particles would cause binding within the mucin layer. Several mechanisms have been documented in literature for the uptake of the nanoparticles/microparticles (Table 9.2).

Table 9.2 Site-specific mechanism for the uptake of the nanoparticles/microparticles
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6.2 Liposomes

Although some success has been achieved in experimental studies involving oral administration of antigens entrapped in liposomes [78], disappointing results have also been reported by others [79, 80]. Nevertheless, a liposomal vaccine has been shown to induce a salivary IgA response in a small number of human volunteers, following oral immunization [81]. Furthermore, although it has been reported that liposomes are unstable in the gut and are not taken up by epithelial cells [82], the uptake of liposomes into Peyer’s patches has been reported [83]. Polymerized liposomes exhibited many characteristics which make them attractive antigen carriers. They are in the nanometer size range suitable to be transported by M cells. Further, liposomes formed by polymerization of 1, 2-bis[(2E,4E)-Octadecadienoyl]-sn-glycero-3-phosphocholine (DODPC) can be manipulated by incorporation of different lipid groups, thus adjusting surface charge and rigidity. DODPC liposomes also have carboxylate groups on their surface which could facilitate cell receptor targeting [84]. A number of studies showed the potential of targeted liposomes in the induction of immune response. Sugimoto et al. [85] and Fukasawa et al. [86] showed that liposomes coated with mannopentose and dipalmitoyl phosphatidylethanolamine (Man5-DPPE) could elicit strong cellular immune responses. It has been shown that UEA1-coated liposomes can be efficiently targeted to murine M cells in vivo [30], thus supporting the hypothesis that decorating liposomes with M cell specific lectins may efficiently target orally delivered antigens to M cells and possibly DCs residing in the FAE.

Liposome offers a number of potential advantages for the mucosal vaccine delivery [87]. While native liposomes may target Peyer’s patch M-cells [83], the efficiency of binding and subsequent uptake is thought to be relatively low following oral gavage of mice [88, 89]. Therefore, we have developed lectin conjugated liposomes for M-cell targeted vaccine delivery [90]. The activity of the liposome-conjugated with UEA-1 towards exogenously provided BSM and affinity toward competing sugar were studied to determine targeting efficacy of lectinized liposomes. The lectinized liposomes showed good BSM binding in absence of specific sugar for UEA-1 (α-l-fucose). The same formulations, however, showed significant decrease in the percent BSM binding in the presence of α-l-fucose (Fig. 9.2). M-cell targeting of the liposomes was studied by dual staining by using CLSM. The interaction of liposomes with M-cells may be facilitated by the relatively thin M-cell glycocalyx which appears to promote the interaction of small particles with the M-cell surface membranes. Also, there are various other factors which may affect the Peyer’s patch uptake of particles [5]. Lectinized liposomes showed higher immune response in comparison to non-lectinized formulation (Figs. 9.3 and 9.4). UEA-1 anchored liposomes selectively targeted to M-cells of Peyer’s patch, and M-cell adherent liposomes are thought to be rapidly endocytosed. M-cell apical surfaces are coated with glycoproteins that display glycosylation patterns different from their enterocyte neighbors, and although the protein backbones have not been identified, the carbohydrate epitopes can be useful M-cell identifiers [91]. Thus, M-cell targeted liposomes were found to have greater accessibility to M-cell and as a consequence they showed enhanced immune response as compared to non-lectinized liposomes.

Fig. 9.2
figure 2

Binding of BSM to lectinized liposomes (EDS3L1-EDS3L5) and optimized non-lectinized liposomes (EDS3) in presence and absence of competing sugar (Reproduced with permission from [90])

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Fig. 9.3
figure 3

Serum anti-HBsAg antibody levels with lectinized liposomal (EDS3L3) and optimized non-lectinized liposomes (EDS3). The animals were immunized orally with the preparation equivalent to the 10 μg HBsAg for three consecutive days and booster dose was given after third week of first immunization. Single intramuscular immunization with booster dose after third week was given with the alum-HBsAg to serve as standard and HBsAg without liposomes was administered orally followed by a booster dose after third week for the comparison. After 4 weeks there was significant difference (P < 0.001) among EDS3L3, EDS3, HBsAg-Oral or HBsAg-IM, however, the difference between EDS3L3 and HBsAg-IM was not significant (P > 0.05). (Reproduced with permission from [90])

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Fig. 9.4
figure 4

sIgA level in mucosal secretions after oral immunization with HBsAg encapsulating lectinized liposomes (EDS3L3) and optimized non-lectinized liposomes (EDS3). The animals were immunized orally with the preparation equivalent to the 10 μg HBsAg for 3 consecutive days and booster dose was given after third week of first immunization. Single intramuscular immunization with booster dose after third week was given with the alum-HBsAg to serve as standard and HBsAg without liposomes was administered orally followed by a booster dose after third week for the comparison. The salivary, intestinal and vaginal secretions were collected after eight week of first immunization. The differences in the antibody level was significant (P < 0.05) among EDS3L3, EDS3, HBsAg-Oral or HBsAg-IM. (Reproduced with permission from [90])

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7 Targeting of Mucosal Vaccine Delivery Systems

Targeting to the specific site of the gastrointestinal tract is an effective means for enhancing the uptake of the particulate systems. Depending on the pharmaceutical application, different targets within the gastrointestinal tract can be exploited (Fig. 9.5) including mucus glycoproteins (mucins), epithelial cells, M-cells, Peyer’s patches or GALT, and abnormal glycoproteins secreted by cancerous cells (local tumors). Brayden et al. reviewed novel M-cell surface receptors that could be used to target orally delivered antigens [34]. Gene expression technology has provided evidence that coculture model has many characteristics of Peyer’s patches. It has been demonstrated that epithelial genes that were unregulated in coculture corresponds to genes expressed selectively in mouse FAE [92]. These include claudin 4, laminin β3, tetraspanTM4SF3 and a matrix metalloproteinase. Claudin 4 appears to have dual location at tight junctions (M cell–enterocyte), and as an M cell and enterocyte cytoplasmic receptor, it is involved in the trafficking of pathogens across M cells to lymphocyte or dendritic cells. Peptidoglycan recognition protein (PGRP)-S and PGRP-L are other potential targets co-localized with UEA-positive cells in microdissected mouse Peyer’s patches and in the FAE respectively [93]. Other targeting agents like lectins, invasins, antibodies, etc. can be used as a means of enhancing targeting and thus in turn particle uptake. Various ligands for the targeted immunization are summarized in Table 9.3.

Fig. 9.5
figure 5

Various options for the targeted delivery. Mucus glycoprotein, M-cells and abnormal glycoprotein secreted by the cancerous cells can serve as receptor for binding with various ligands conjugated to drug/vaccine delivery system

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Table 9.3 Various ligands for targeted mucosal immunization
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7.1 Lectin Mediated Targeting

Lectins are proteins or glycoproteins capable of specific recognition of and reversible binding to carbohydrate determinants of complex glycoconjugates, without altering the covalent structure of any of the recognized glycosyl ligands. Lectin receptors are expressed on various cells such as endothelial cells, hepatocytes, macrophages, monocytes and lymphocytes. They are efficient in recognizing the complex oligosaccharide epitopes, which are also present on the cell surface or could be exogenous glycoconjugate ligands mimics of endogenous carbohydrate epitopes [104]. Lectins are potential tools for the targeting of particulate vaccines to the M cell of the Peyer’s patches, which are the sampling site of the mucosal immune system. Nanoparticles/microparticles may also be targeted to mouse Peyer’s patch M cells by coating with the lectin UEA1 for the development of an effective mucosally targeted vaccines. In studies reported by Foster et al. [56], polystyrene microparticles (0.5-mm diameter) were covalently coated with the lectin UEA1 and administered to mice both by injection into ligated gut loops of anaesthetized animals and by oral gavage. In contrast to other proteins, lectin UEA1 coating selectively targeted the microparticles to mouse Peyer’s patch M cells, and M cell adherent microparticles were rapidly endocytosed. Although the lectins specific for the human intestinal M cells await identification, human M cells preferentially display the sialyl Lewis A antigen [26] and this could be envisaged for targeting vaccines to the mucosal immune system. Future studies should determine whether lectins may similarly be used to target vaccine candidate in PLA/PLGA based delivery construct to intestinal M cells, and whether such targeting enhances the immune response to antigens delivered by these carrier systems. Recently our group has developed biodegradable polymer based stabilized microparticles and nanoparticles for the mucosal vaccination [39] and also envisaged lectin for the targeted mucosal immunization [40]. Additionally we have also explored cholera toxin B subunit conjugated bilosomes [43] and mannosylated niosomes [101] as potential carrier-adjuvants for the targeted oral mucosal immunization.

7.2 Invasin Mediated Uptake

Invasins are virulent factor usually associated with the bacterial cell wall, and have the capacity to stimulate cytokine synthesis and to interact with mammalian cells by distinct mechanisms [105]. Young et al. demonstrate the potential of invasins for the internalization process [106]. For this purpose, microparticles were coated with Yersinia enterocolitica invasin and the resulting conjugates put in contact with human laryngeal epithelial cells (Hep-2 cells). The presence or absence of internalised conjugates was monitored by transmission electron microscopy and light microscopy. It was clear that conjugates not only bound, but also were internalised by the Hep-2 cells. In contrast, control conjugates were rarely associated with these cells. Salmonella typhimurium selectively bind to, invade and destroy murine M cells and have been studied as live oral vaccine delivery vehicles. The M cell targeting by S. typhimurium is mediated by a specific adhesin (long polar fimbria; LPF) [107]. Reovirus type 1 is another ligand, which selectively adhere to, and endocytosed by intestinal M cells. It was demonstrated that proteolytic processing of native reovirus type 1 is required for adhesion to murin M cells and this is dependent on retention of modified σ1 and/or product of μ1 outer capsid protein. It has been suggested that σ1 protein has potential for targeted delivery [108].

7.3 Antibody Directed Targeting

The use of antibodies and monoclonal antibodies has been proposed for specific targeting within the gastrointestinal tract. It was observed that binding of the 5B11 monoclonal antibody, with specificity for rabbit M cells, to polystyrene particles, enhanced uptake by rabbit M-cells 3–3.5-times when compared to controls (plain latex and IgM of unrelated specificity-conjugates) [109]. The ability of different conjugates, obtained by coating latex microparticles with albumin, bovine growth hormone (bGH), human IgG, secretory IgA (hIgA), and bGH complexed with an IgG antibody raised against bGH (bGH-Ab), to be taken up by M cells was studied. It was found that the selectivity in binding to and entry into M-cells was improved by the use of IgG or bGH-Ab. Moreover, the appearance of conjugates in rat mesenteric lymph showed a similar selectivity to that found for binding and entry into M-cells. Ferritin-loaded liposomes conjugated to IgA were investigated for mucosal immunization via the rectum [109]. It was observed that the presence of IgA on the liposome surface increased the uptake of conjugates by Peyer’s patches, and the local rectal/colon immune response to ferritin about fivefold over uncoated liposomes.

8 Link Between M Cell Uptake of Particles and Induction of Mucosal Immunity

Generally, macromolecules that adhere to mucosal surfaces tend to induce vigorous mucosal immune responses, whereas soluble, non-adherent proteins do not [110]. Pathogens and vaccines that can bind selectively to M cells appear to be most effective in mucosal invasion and induction of mucosal immune responses, and this assumption underlies many of the current approaches to vaccine design. Presently, there have been few successful oral vaccine trials in man using non-live antigens in particles. Mixed results were obtained in a limited number of human subjects dosed with untargeted PLG microspheres containing the highly potent E. coli colonization factor antigen II as potential vaccine for enterotoxigenic E. coli [111]. These studies suggest indirectly that there is likely to be some particle uptake in man; however, a quantifiable relationship between enhanced M-cell targeting of vaccine loaded particles and an enhanced immune outcome remains illusive. Apart from the immunology issues, pharmaceutical factors are critical in the design of antigen-loaded particles. These include antigen stability issues, premature antigen release from particles in the intestine and incomplete antigen release within Peyer’s patch at the right time. There are many reports of induction of immune response using M cell targeted particles in animal models; however, more clinical studies are required to establish a correlation between M cell targeting and elicitation of immune response. A Phase I trial of a single shot tetanus toxoid (TT) and diphtheria toxoid in PLGA microspheres is still some way off, even though outcomes in mice and guinea-pigs showed positive and durable immune responses using antigen-loaded microparticles [54]. It is possible that particulate antigens, when targeted to inductive immune sites, might perform even better in man than in laboratory animals because the pathways of antigen presentation by human dendritic cells are relatively well established and might even be superior [112]. Advancement in the non-living vaccine delivery system coupled with the suitable targeting strategy may lead to a successful clinical trial.

9 Future Perspectives

Recent discoveries in both mucosal vaccine delivery and mucosal adjuvant research have significantly improved the effectiveness of mucosal immunization in animal models. The mucosal immune system is a complex system that generate large amount of s-IgA as well as cell mediated immunity at mucosal surfaces to prevent pathogen infiltration and inflammation. The mucosal immune system should be most efficient in providing protection against pathogens and generating longer lasting protection through using attenuated pathogen for vaccine. The only mucosal vaccines approved for humans are attenuated pathogens. Future mucosal vaccines will also involve vaccines strategies other than attenuated pathogens. New delivery strategies such as immunization of live recombinant vectors, DNA plasmids, and transgenic plants to deliver antigens present promises to improve the efficiency of mucosal antigen delivery. Further, DNA vaccines and subunit vaccines such as bacterial adhesion in combination with potent mucosal adjuvants (such as QS21; a saponin, unmethylated CpG motifs or cytokines such as IL-12) or mucosal delivery systems based on nanoparticles/microparticles will have the potential to be the next generation of vaccines.

Mucosal delivery of vaccines offers a number of significant advantages over systemic delivery. There are many alternative approaches to the mucosal delivery of vaccines and our group has explored various versions of delivery systems [39, 40, 43, 90, 101, 113]. One potential approach to the mucosal delivery of vaccines is the encapsulation or entrapment of antigens into polymer based nanoparticles or microparticles. Polymeric delivery systems can be manipulated to enhance the efficacy of mucosally administered vaccines in a number of ways; they can protect antigens from degradation, concentrate them in one area of the mucosal tissue for better absorption, extend their residence time in the body, or target them to sites of antigen uptake (e.g., Peyer’s patches in the gut).

Immunization does not always stimulate immunity because of the insufficient elicitation of immune responses. Such limitations have spurred the development of new adjuvant and antigen-delivery systems. Adjuvant plays an important role in enhancing the efficacy of vaccines. Recombinant proteins or synthetic peptides are safer than crude inactivated microorganism, but less immunogenic. This limitation can be overcome by using specific adjuvant. The adjuvant selection depends on several criteria, like the target species, the antigens, the type of desired immune response, the route of administration, or the duration of immunity. So far, biodegradable polymers particularly of PLGA have been used, considerably because of their well-known degradation properties. An area requiring additional efforts is analytical characterization of protein-encapsulated nanoparticles/microparticles. Advanced methods for protein characterization is in demand to approach problem of protein stabilization in polymer based delivery systems. Development of in vitro–in vivo correlation for protein release from protein nanoparticles/microparticles is another issue. More intensive interactions between immunologists and drug delivery specialists are required to understand protein release and its presentation to the immune system. Significant progress has been made recently with biodegradable polymer, mainly PLGA and various approaches are being considered for the effective stabilization of proteins in microparticles during the preparation process. Among them we have focused on the basic additives mediated stabilization of therapeutic protein within carrier construct [39, 40]. Nevertheless, all the approaches involving encapsulation of antigens into nanoparticles/microparticles are likely to suffer from the some significant drawback; the extent of uptake of the particles across the gut appears to be limited. Whether or not the extent of uptake in humans is sufficient to allow the development of an effective oral vaccine is currently unknown. However, it is clear that in rodents, the extent of uptake of nanoparticles/microparticles can be enhanced using targeting ligands.

Numerous studies suggest that the efficiency of particle absorption can be improved through modification of particle surfaces with targeting molecules such as antibodies or lectins. Albeit the results are promising, it is not known if any of the strategies will be effective in humans because it is currently unclear if particulates are taken up in human GIT. Additionally the uptake mechanisms and absorption efficiencies are not known. Thus, current knowledge obtained from animal models may or may not be extendible to human beings. Continued research to understand the interconnection and sub-compartmentalization of the common mucosal system will certainly guide the rational selection for routes of mucosal administration. An efficient delivery vehicle, combined with an effective adjuvant given through an optimal route of administration, will ultimately allow for the development of a successful needle-free (mucosal) vaccine in humans.