Oral delivery of nanoparticle-based vaccines.

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Oral delivery of nanoparticlebased vaccines Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/30/14 For personal use only.

Expert Rev. Vaccines Early online, 1–16 (2014)

Nirmal Marasini1, Mariusz Skwarczynski1 and Istvan Toth*1,2 1 School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia 2 School of Pharmacy, The University of Queensland, Woolloongabba, QLD 4102, Australia *Author for correspondence: Tel.: +61 733 469 892 Fax: +61 733 654 273 [email protected]

Most infectious diseases are caused by pathogenic infiltrations from the mucosal tract. Therefore, vaccines delivered to the mucosal tissues can mimic natural infections and provide protection at the first site of infection. Thus, mucosal, especially, oral delivery is becoming the most preferred mode of vaccination. However, oral vaccines have to overcome several barriers such as the extremely low pH of the stomach, the presence of proteolytic enzymes and bile salts as well as low permeability in the intestine. Several formulations based on nanoparticle strategies are currently being explored to prepare stable oral vaccine formulations. This review briefly discusses several molecular mechanisms involved in intestinal immune cell activation and various aspects of oral nanoparticle-based vaccine design that should be considered for improved mucosal and systemic immune responses. KEYWORDS: adjuvant • bilosomes • liposomes • M-cells • mucosal vaccine • nanoparticle-based vaccine • oral vaccine • virus-like particle

Oral delivery of vaccines

Infection remains a major cause of human mortality. In the last few decades, vaccines have been the greatest triumph in the medical field and have saved more lives than any other available drugs. Vaccination serves as the best prophylactic approach for combating infectious diseases owing to their efficacy and costeffectiveness. Vaccines are composed of antigens, which may be live-attenuated, inactivated, killed organisms or minimal fractions of pathogenic organisms including proteins or peptides responsible for producing the desired immune response against infections. Most pathogens (~90%) invade the body tissues via the mucosal route, through gastrointestinal (GI), respiratory and urogenital systems. Body mucosal systems are more vulnerable to pathogens due to their large exposed area and in contrast to skin, mucosal membrane barriers are thin and permeable. Thus, as a first-line of defense, an effective mucosal immune response is desirable to fully protect against pathogens and actions of their toxins at the mucosal surface. Secretory IgA (SIgA) is a major immunoglobulin, which plays a key role against pathogen invasion at the mucosal sites [1]. Mucosal immunity gained at one site has been shown to offer protection to other remote mucosal sites by the common mucosal defense informahealthcare.com

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network in the body [2]. However, the mucosal immunity at distal sites may be comparatively lower than the immunity gained at the primary effector site. To date, the majority of the currently available vaccines are administered by invasive, inconvenient or unpleasant routes using injections that may result in low patient compliance. Injectable vaccines (either subcutaneous or intramuscular) have high potential to produce robust and effective immune responses both at cellular and systemic levels to prevent the disease, but are not effective in preventing infections at mucosal entry sites due to insufficient production of specific mucosal immune responses. These shortcomings associated with injectable vaccines have attracted the interest of a wide range of researchers from the biopharmaceutical industry and academia to develop an alternative needle-free oral vaccine delivery system. Oral vaccine delivery has several advantages such as improved patient compliance, capacity for mass immunization, selfdelivery or easy administration, simplified production and storage, low production cost and no needle-associated risks (injuries and infections). In addition, oral vaccines are superior to injectable vaccines due to their ability to produce both antigenic-specific systemic antibodies (IgG) in blood and mucosal antigenspecific (IgA) antibodies. Oral vaccines need to

2014 Informa UK Ltd

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Marasini, Skwarczynski & Toth

Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/30/14 For personal use only.

Table 1. Oral mucosal vaccines (licensed and under clinical trial)

[125–127].

Brand

Infection/disease

Antigen (s)

Formulation

Stage

Dukoral

Cholera

Inactivated Vibrio cholera with CTB-subunit

Suspension

Market

Ardovax

Febrile acute respiratory disease

Adenovirus Type 4 and Type 7 vaccine, live

Enteric coated tablets

Market licensed only for military population (17–50 years age)

Rotarix

Gastroenteritis

Live-attenuated monovalent human rotavirus RIX4414 strain of G1P[8] type

Suspension

Market

RotaTeq

Gastroenteritis

Attenuated pentavalent live rotavirus reassortants: G1, G2, G3, G4 and P1A[8] derived from human and bovine species

Solution

Market

Vivotif

Typhoid

Live-attenuated strain of Salmonella typhi Ty21a

Enteric-coated capsule

Market

Vario

Polio

Live-attenuated trivalent OPV vaccine sabin strains 1, 2, 3

Liquid

Market

PXVX0103

Bird flu (avian influenza)

Live adenoviral-based vaccine (Ad4-H5-Vtn) against avian influenza (H5N1)

Capsules

Phase I clinical trial completed

N/A

HIV

Live, adenovirus-based vectors, (Ad4-env Clade C and Ad4-mGag)

N/A

Preclinical stage/NDA approved by US FDA

PXVX0200

Cholera

Live-attenuated oral cholera vaccine strain CVD 103-HgR

N/A

Phase II clinical trial completed

CTB: Cholera toxin B; N/A: Not available; NDA: New drug application; OPV: Oral poliovirus.

be exposed to the gut, which is already full of commensal bacteria, enzymes, nutrients and lumens. Thus, in terms of the scaled-up and manufacturing perspective, oral vaccines do not require a high degree of purity in contrast to their injectable counterpart. However, injectable vaccines are required to be highly pure and completely free from endotoxins [3]. Currently, most of the licensed oral vaccines are prepared from robust technologies using either live-attenuated or killed microorganisms [4]. The recent progress in clinical oral vaccine development is shown in TABLE 1. Nevertheless, the oral route, being the most preferred, comes with numerous hurdles for effective delivery to the immune cells. First, an oral vaccine has to suffer exposure to an extremely acidic pH, proteolytic enzymes and bile salts leading to its degradation in the gastrointestinal tract (GIT). Second, vaccines need to overcome various biological barriers (e.g., presence of tight epithelial cellular junctions and a thick mucous layer) in the intestinal lumen for effective uptake and permeation across the intestinal walls. In addition, lower uptake of the antigenic particles is also due to the short antigenic exposure time to mucosal tissues. Thus, for a potent and longlasting immunogenic effect, oral vaccines may require larger and multiple numbers of doses compared with their systemic counterparts [5]. However, repetitive oral administration of larger oral doses of antigens might induce systemic nonresponsiveness (oral tolerance) [6]. The lack of availability of potent immunostimulants or mucosal adjuvants with low toxicity is another major hindrance in the development of oral vaccines [7]. Furthermore, the major constraints of oral vaccines doi: 10.1586/14760584.2014.936852

are difficulties of quantification of the actual degree of immune response, particularly IgA, at different mucosal sites following oral administration. Recently, Saletti et al. described a protocol for quantification of SIgA in blood circulation based on specific mucosal antibody-secreting cells. They measured expression of mucosal integrin a4b7 which coexpress chemokine receptors (CCR9 and CCR10) that are able to produce polymeric IgA, a precursor of SIgA [8]. Each type of antigen behaves in a different way and there is not a single technological platform to deliver all types of vaccines. Encapsulating antigenic material using several polymeric and lipid-based nanoparticle (NP) carriers could be an effective approach as such particles can reduce degradation of antigens in the GI system. NP systems themselves possess some degree of intrinsic adjuvant or immunostimulant properties and also have the ability to co-encapsulate multiple antigenic epitopes, targeting ligands and external adjuvants into a single carrier. An ideal vaccine carrier is expected to protect the structural integrity of the antigen and effectively deliver it to the desired mucosal surface in order to produce sufficient mucosal, humoral and cellular responses (BOX 1). The formulation of an ideal vaccine requires a thorough understanding of molecular and cellular components of GI physiology. This review briefly discusses the physiological considerations of oral vaccine design and various NP-based formulation approaches for oral vaccine delivery. Intestinal immune system

The human intestine is composed of tightly adhered epithelial cells, which maintain immune homeostasis by distinguishing Expert Rev. Vaccines


Oral delivery of NP-based vaccines

between harmful, foreign microorganisms and harmless nutrients or commensal organisms. The intestine houses the highest number of immune cells compared with other mucosal systems. The intestinal immune system is regulated by gutassociated lymphoid tissue (GALT), which contains both inductive and effectors sites (FIGURE 1). Inductive sites are located in the intestinal tissues where antigens are encountered. Major inductive sites in the gut include organized Peyer’s patches (PPs), lymphoid follicles and scattered antigen-presenting cells (APCs) in the intestinal epithelium and the vermiform appendix. The antigens from the intestinal lumen are taken up by local lymphocytes into inductive tissues that differentiate to produce effector actions (antibody production, cytokine secretions and cytotoxicity) and migrate to the effector tissues via the lymphatic system. The major effector sites in the intestine are the lamina propria (LP) and surface epithelium. PPs consist of highly organized immune cells in the GALT [9]. Anatomically, PPs are dome-shaped, slightly elevated lymphatic organs that reside mostly in the mucosal region throughout the ileum. They are covered by a single layer of specialized epithelial cells, the follicle-associated epithelium (FAE). PPs extend up to the submucosal layer, which contains 10–1000 aggregated lymphoid follicles [10]. Antigen sampling & their transport by immune cells in the intestine

The first and crucial moment for inducing immune responses is efficient antigen sampling from the luminal region of the intestine. However, the intestine has a protective barrier formed by a layer of epithelial cells, which selectively permit permeation of luminal antigens and microbes. Goblet cells secrete mucus, which forms a hydrated gel-like film at the surface of epithelium and avoid direct contact of foreign materials with epithelial cells. They are more numerous in the large intestine than in the small intestine. The mucus of the small intestine is one-layered and loosely attached to the epithelium that makes them more permeable to the foreign antigens. Therefore, the majority of intestinal immune system is active in the small intestine. In contrast to small intestine, the mucus of the large intestine is thicker and organized into two layers, the inner layer being less permeable [11]. The schematic representations for transport of various antigens are summarized in FIGURE 2. Microfold-cells (M-cells) express a wide range of M-cell-specific receptors on their apical surface in the luminal region, which are involved in phagocytosis of the antigens or pathogens (FIGURE 2A) [12]. Some examples of endocytic receptors typically expressed in M-cells are GP 2, ganglioside (GM1), b1 integrins, cellular prion protein and C5a receptors, which can be used for M-cell targeted oral vaccine delivery [13]. M-cells are specialized immune cells of the GALT that lie within the FAE that transport macromolecules, particles and microorganisms from their apical surface to the basolateral membrane [14]. Mostly M-cells have thin mucous layers devoid of or having shorter microvilli, which differentiate them from other intestinal epithelial cells. M-cells internalize particles by various mechanisms such as endocytosis of clathrin-coated informahealthcare.com

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Box 1. Properties of an ideal oral vaccine. • Low cost • Easy administration without medical professionals and

special devices • Capacity for large-scale production • Stability under lyophilization and avoidance of cold-chain

storage • Sufficient protection of antigens against gastrointestinal

fluids (proteolytic enzymes, low pH and bile salts) • High antigen loading/encapsulating capacity of particles • Strong mucosal adjuvanticity • Prolonged exposure of antigens to antigen-presenting cells • Optimum size for effective transportation of particles across

intestinal lumen • Sufficient targeting ability to intestinal cells (microfold-cells) • Produce long-term mucosal and systemic immunity • Adequate safety profile

vesicles, actin-dependent phagocytosis or macropinocytosis [15]. They are capable of ingesting particles from nanometers to a few micrometers (1–5 mm) [16]. These unique features make M-cells a promising target for delivery of oral vaccines [17]. The M-cells in the intestinal tract cover less than 1% of the total absorptive surface area of epithelial cells [18]. It has been reported that Mcells in the FAE makeup approximately 5 and 10% of total epithelial cells in humans and mice, respectively [19]. In addition, the density and function of M-cells in the intestine has been shown to be age-dependent. In a study in aged mice, the functional M-cell marker (Ets transcription factor Spi-B and chemokine ligand 20) expressions were dramatically reduced suggesting the decreased M-cells maturation capacity in aged mice [20]. Besides, aged mice showed reduced capacity to transcytose the particulate luminal antigens across the FAE. The result suggests that the efficiency of M-cells-targeted vaccines could be reduced and may not produce the same or comparable immune responses in the elderly. The subepithelial LP region of GALT contains two main populations of intestinal mononuclear phagocytes, compromising dendritic cells (DCs) and macrophages. The CX3CR1+ mononuclear phagocytes and CD103+ DCs are the major population of mononuclear phagocytes in the intestinal LP. The former are tissue resident macrophages, whereas later are considered as migratory DCs. The CX3CR1+ macrophages uptake antigens and transfer to CD103+ DCs [21]. The morphology of DCs in the GIT is unique compared with other DCs due to their ability to extend their projections transepithelially across the enterocytes and M-cells for luminal sampling of antigens (FIGURE 2B & 2C). Targeting intestinal DCs is a promising strategy for effective antigenic delivery in the development of novel oral vaccines. Antigens can be conjugated to DCs targeting peptides for its efficient uptake by DCs. For example, antigenic probiotic vector (Lactobacillus) species conjugated to DCs targeting peptide (FYPSYHSTPQRP) showed strong doi: 10.1586/14760584.2014.936852

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Inductive sites A

Naive CD4+ T-cells D

Particulate antigens Naive CD4+ T-cells Primed CD4+ T-cells B-cells IgA+ B-cells Polymeric Ig receptor (pIgR) Dendritic cells SIgA IgA+ plasma cells

B


Peyer's patch

Primed CD4+ T-cells Intestinal lumen Effector sites SIgA

IgA+ B-cells B-cells

pIgR

Blood drainage Dimeric IgA

C Lamina propria Peripheral lymph node

IgA+ plasma cells Afferent lymphs

antigens [24]. For example, neonatal Fc receptors (FcRn) are expressed in enterocytes (FIGURE 2E). Selective targeting to FcRn receptors in enterocytes allows effective internalization of antigens across the epithelial barrier to be presented to the DCs. Pridgen et al. showed that oral administered NPs modified with IgG Fc selectively targeted FcRn receptor and crossed epithelial barrier 11-fold more efficiently than unmodified particles [25]. In addition, the particles can also be transported by transcellular process mediated by absorptive enterocytes and paracellularly in between the enterocytes (via tight junctions) (FIGURE 2F & 2G) [26]. These particulate antigens can also gain direct access to blood circulation (FIGURE 1D). Thus, the integration of several passive and active transport systems for particle transportation across intestinal cells (M-cells and enterocytes) is responsible for effective induction of an immune response.

IgA+ B-cells Mesentric lymph node

Mucosal immune response after oral vaccine administration

Following oral immunization with particulate vaccines, antigens are sampled by specialized M-cells, DC projections or Systemic circulation enterocytes and are delivered into underlying APCs in PPs (FIGURE 1). Then, antiFigure 1. Intestinal immune system and immune responses after administration of particulate vaccine. (A) After particulate antigens are taken up at the inductive gens are often internalized and processed sites, they are presented to DCs. These antigen-loaded DCs prime naı¨ve CD4+ T-cells in into small fragments by DCs (FIGURE 1A). + the PPs. The primed CD4 T-cells in turn trigger B-cells and undergo isotype switching to These antigen-loaded DCs provide cogenerate antigen-specific IgA+ B-cells. These IgA+ B-cells leave the PPs through afferent stimulatory signals to naı¨ve CD4+ T-cells. lymph to mesenteric lymph node and finally reach the blood circulation. (B) Similarly, The primed CD4+ T-cells further react antigen-presenting DCs actively migrate to the mesenteric lymph nodes for further CD4+ + + T-cell activation and subsequent IgA B-cell production. The IgA B-cells further leave with antigen-specific B-cells and undergo mesenteric lymph nodes to the blood circulation. The circulating antigen-specific IgA+ class-switching to IgA+ B-cells. Then, B-cells move to distant effector sites in the LP and undergo differentiation and IgA+ B-cells leave PPs through afferent maturation to generate high-affinity IgA+ producing plasma cells (enhanced by cytokines lymphatics to the regional mesenteric IL-5 and IL-6, subsets of Th2 cells), which in turn produce dimeric or polymeric forms of lymph node before reaching the systemic IgA. (C) The dimeric or polymeric IgA binds to Ig receptors expressed on the basolateral surface of epithelial cells to form SIgA. (D) This complex is further transcytosed toward blood circulation. Similarly, antigenthe luminal surface of the intestine (effector sites). Alternatively, particulate antigens presenting DCs actively migrate to lymph directly reach the systemic circulation from the gut and interact with T-cells in the nodes for further CD4+ T-cell activation peripheral lymph nodes. and subsequent IgA+ B-cell DCs: Dendritic cells; LP: Lamina propria; PPs: Peyer’s patches; SIgA: Secretory IgA. production (FIGURE 1B). These IgA+ B-cells in the MLN are also drained to the interactions with intestinal DCs to produce various humoral blood circulation. Finally, the circulating antigen-specific IgA+ and T-cell-mediated immune responses [22]. Small intestinal B-cells move to distant effector sites in the LP and undergo difgoblet cells were found to be effective for delivering low molec- ferentiation and maturation to generate high-affinity IgA+-proular weight soluble antigens from the luminal region of the ducing plasma cells (process is enhanced by cytokines IL-5 and intestine to underlying LP-based CD103+DCs (FIGURE 2D) [23]. IL-6, subset of Th2 cells) and to produce the dimeric or polyApart from M-cells-mediated sampling, enterocytes also meric form of IgA (FIGURE 1C). The dimeric or polymeric IgA express several receptors that specifically transcytose the binds to polymeric Ig receptors expressed in the basolateral Efferent lymph

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Goblet-cell

M-cell


FcRn

E Enterocytes

A

B D

G C

F

CD103+

Figure 2. Schematic representation for transport of nanoparticulate vaccines across the luminal region of the intestine. (A) Phagocytosis by indwelling macrophages in the M-cell. (B) DC-mediated luminal sampling from the M-cells. (C) Transepithelial DC extension into the luminal region of the intestine. (D) Goblet cell-mediated transport of low molecular weight soluble antigens to CD103+ DCs. (E) Enterocytic receptor-mediated transport (FcRn receptor). (F) Transcellular transport across the enterocytes. (G) Paracellular transport between the walls of enterocytes. DCs: Dendritic cells; M-cell: Microfold-cell.

surface of epithelial cells to form SIgA and further translocates toward the luminal surface of the intestine. The SIgA in the intestinal mucosa is responsible for frontline protection against invading pathogens [27,28]. Influence of particle size in intestinal sampling

Just a simple delivery of antigen and adjuvants to the intestinal lumen does not guarantee production of a complete set of immune responses. The antigens must be able to penetrate the intestinal mucosal layer to initiate an immune response. The antigens can be encapsulated, adsorbed or cross-linked by the polymers that convert them into particles for more efficient immune-modulating capacity than soluble antigens alone [29]. The particulate formulation of antigens has been shown to be less susceptible to oral tolerance than soluble antigens [30]. One of the most important factors that should be considered for transportation across the intestine is the size of the immunogens. Nanometer-sized particles are better taken up by both Mcells and enterocytes. In addition, M-cells and enterocytes also have the capacity to transport particles up to few micrometers (1–5 mm) in size. The small particles can be delivered directly to the systemic immune system (peripheral lymph nodes) via enterocytes to initiate an IgG response, thus lowering the mucosal IgA response [9]. Wang et al. measured the influence of different-sized particles (130 nm, 430 nm and 1–2 mm) loaded with model antigen bovine serum albumin in generating mucosal and systemic antibodies after oral administration [31]. Higher immune responses (IgA and IgG) were obtained for the small informahealthcare.com

particles (130 and 430 nm) compared with larger particles (1– 2 mm). Thus, they concluded that particles below 500 nm are better taken up by M-cells and enterocytes than larger particles (1–2 mm). On the contrary, Gutierro et al. reported a higher IgG titer obtained with 1 mm particles compared with 200 and 500 nm particles [32]. Awaad et al. measured PPs’ uptake efficiency using single-sized (95, 130, 200, 340, 695 and 1050 nm) particles [26]. They suggested that a particle size ranging from 95 to 200 nm was an optimal size for efficient vaccine delivery rather than a larger particle size (>300 nm). The findings suggest that the optimum particle size for oral vaccine remains controversial. In fact, it will be interesting to use a mixture of polydisperse-sized particles (below 2 mm) for effective induction of systemic and mucosal immune responses. NP-based oral vaccine delivery system

A good oral vaccine candidate should adequately protect against the hostile environment of the GIT and deliver sufficient antigens to the targeted site to produce the required protection against the pathogens. With the advancement in proteomics and high-throughput screening in chemical biology, antigens associated with the disease can be easily identified. To date, most oral vaccines are delivered as live-attenuated/inactivated microorganisms combined with adjuvants. Such microorganism might reverse to virulent state (especially in immunocompromised patients) that replicate inside the GI epithelium ultimately harming the host. Other disadvantages include associated adverse reactions (autoimmune and allergic doi: 10.1586/14760584.2014.936852

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Aqueous interior with hydrophilic antigens

Phospholipid bilayer encapsulating hydrophobic antigens A Liposomes W/O

O/W

Lipophilic antigens in oily core


Oral vaccines Hydrophilic antigens in aqueous core

Gastric barriers 1. Low pH (1-4) 2. Proteolytic enzymes

O/W/O W/O/W B Emulsions

Lipophilic antigens Nanoparticulate delivery system

Quil A saponin

C ISCOMs Foreign epitopes

Intestinal barriers 1. Bile salts 2. Thick mucus layer 3. Tight epithelial cell junctions 4. Limited permeability for a. Too hydrophilic and b. High molecular weight antigens

D Virus-like particles Antigens conjugated, adsorbed or encapsulated

E Polymeric nanoparticle Antigens conjugated or adsorbed

F Inorganic nanoparticles

Figure 3. Various barriers in the gastrointestinal system encountered by oral vaccines and nanoparticulate vaccine delivery strategies to overcome these problems. o/w: Oil in water; o/w/o: Oil in water in oil; w/o: Water in oil; w/o/w: Water in oil in water.

responses) and difficulties in pathogen culturing. A subunit vaccine is an alternative approach to overcome these disadvantages [33]. They are composed of minimal fractions of structural components of target pathogens such as protein antigens and polysaccharides. However, subunit oral vaccines are of limited use owing to low oral stability and lower immunogenicity. Efforts are currently being directed towards increasing immunogenicity of the subunit vaccines by attaching adjuvants (e.g., doi: 10.1586/14760584.2014.936852

lipids) and delivering them using appropriate particulate carriers [34,35]. Recently, NP-based vaccine delivery has been gaining popularity due to its ability to co-deliver antigens and adjuvants in a single particulate carrier [36,37]. NPs have shown more potent immunostimulatory capacity than conventional aluminumbased salts (alum) as adjuvants and may not require the addition of APC targeting ligands to potentiate immunogens [38,39]. Expert Rev. Vaccines


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Table 2. Pros and cons of oral nanoparticle carriers. Nanoparticle system

Advantages

Disadvantages

Nanoliposomes

• Phospholipids possess intrinsic adjuvant property • Can incorporate both hydrophilic and lipophilic antigens • Modified liposomes such as bilosomes or arachesomes are relatively stable in GI fluids

• Limited antigen loading, especially hydrophilic antigens • Naked liposomes have poor GI stability • Weaker mucus penetrating property

Nanoemulsion

• Self-adjuvant • Can encapsulate both hydrophilic and lipophilic antigens • Inclusion of surfactant can provide better permeation across intestine

• Chances of premature antigen leakage • Poor GI stability

ISCOMS

• Potent built-in adjuvant (Quil A) • Easy encapsulation of lipophilic antigens

• Do not exert depot release profile (rapidly cleared) • Difficult to incorporate hydrophilic antigens

Virus-like particles

• Mimics original virus • Self-adjuvant • Higher GI stability

• Polydispersed particle size • Lack of reproducibility in batch-to-batch production

Polymeric NPs

• Surface properties can be easily tailored for better immunogenicity • Release antigen at targeted sites

• • • • •

Inorganic NPs

• Better protection of adsorbed antigens • Less chances of premature release • Easy surface modification

• Low biodegradability • Lesser information on role as adjuvants • Poor aqueous solubility

Limited aqueous solubility Premature (burst) release Insufficient antigen protection Low antigen loading NPs synthesis might require use of organic solvents

GI: Gastrointestinal; ISCOMS: Immunostimulating complexes; NPs: Nanoparticles.

NPs can be engineered to encapsulate vaccine components inside or attached to their surfaces for efficient presentation to APCs. Besides, NPs offer several unique controllable properties (which influence their immunogenicity) such as their particle size, surface properties (charge, surface area), high loading efficiency, sufficient protection in gastric fluid and enhanced penetration capacity across the mucosal barrier of the intestine. NPbased carriers can be decorated with functional molecules to target the immune cells, enhance stability and modify the release property of antigens. In this review, NP-based oral vaccine delivery system was categorized in following groups: lipids, polymers and vectorbased systems (liposomes, emulsion, immunostimulating complexes [ISCOMs], polymeric NPs, inorganic NPs and vector-mediated virus-like particles [VLPs]) (FIGURE 3). There are clear merits and demerits of using each NP as oral vaccine carriers. The pros and cons of different NPs formulation strategies are summarized in TABLE 2. Nanoliposomes

Liposome vesicles have been well-documented in the literature for their diverse ability to deliver various hydrophilic and lipophilic antigens [40]. Liposomes are closed self-assembled structures of phospholipids consisting of an internal aqueous core entrapped by a lipid bilayer. The internal core can effectively encapsulate hydrophilic antigens, whereas the lipophilic informahealthcare.com

antigens can be entrapped, attached or adsorbed onto the phospholipid bilayer or at the interface of an aqueous core and phospholipid layer. The particulate nature of liposomes helps to encapsulate a broad range of immunomodulators and antigens within its core, and to provide prolonged release and specific uptake by immune cells. These unique features make liposomes an efficient tool for delivering antigens and immune potentiators within a single particulate carrier [41]. The surface properties of liposomes can be easily modified to increase encapsulation efficiency, stability, slow release, mucosal adherence and immune cell targeting capacity [42]. Despite several advantageous features of liposomes, they have limitations such as low antigen loading and poor stability. Conventional liposomes are unstable in gastric juice and easily digested by pancreatic lipase [43]. Also, intestinal bile salts can disrupt the phospholipid membrane integrity and lyse the liposomes resulting in the premature release of antigens. Unfortunately, the degree of lipolysis of phospholipids in the GIT is difficult to measure in vivo, so in vitro models are often used. For example, Parmentier et al. measured the stability of the two different formulations containing hydrated egg phosphophatidylcholine and soy phosphatidylcholine [44]. The rate and extent of hydrolysis by pancreatic enzymes was studied. They concluded that hydrated egg phosphophatidylcholine was more stable with only 30% digestion of phospholipids compared with soy phosphatidylcholine with 80% digestion after 1 h incubation. There doi: 10.1586/14760584.2014.936852


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was no direct relationship between vesicle size and phospholipid digestion [44]. The stability of liposomes can be altered by changes in their composition. For example, the inclusion of cholesterol in the liposomes provides more rigid and stable liposomes. The stability of retinol-encapsulated liposomes was significantly increased with the increase in cholesterol concentration of the liposomes (with the maximum stability at 50% cholesterol content) [45]. Phospholipids with the higher phase transition temperature are more stable to the body temperature. Formulating liposomal mucosal vaccines are mostly benefited by using the lipids that have slightly higher transition temperature than the body temperature because at this temperature, lipids are lesser rigid and provide better mucosal adherence [42]. To cope with stability issues in the GIT, liposomes can be formulated as bilosomes by modifying their formulation. Bilosomes are liposomes that have incorporated biodegradable and biocompatible bile salts (e.g., sodium deoxycholate) [46]. The incorporation of bile salt stabilizes liposome vesicles against destructive effects mediated by bile salts present in the GIT. Besides improving the stability of bilosomes, sodium glycocholate itself acts as membrane permeation enhancer in liposome formulations due to its increased fluidity in the intestinal tract [47]. An in vitro stability study of bilosomes did not show any remarkable loss of antigen in the simulated gastric fluid, whereas slow antigen release was observed in the simulated intestinal fluid [48]. Furthermore, when bilosomes were administered orally, they efficiently induced a mucosal IgA response not only at the site of induction, but also at other distant mucosal sites [49]. Bilosomes were able to be specifically taken up by M-cells eliminating requirements for a larger dose of antigen, thereby reducing the risk of oral tolerance. A double dose of orally administered bilosomes and a single dose of intramuscularly administered hepatitis B antigen induced similar IgG antibody production [50]. Bilosome size plays a significant role in immune responses. It has been widely accepted that compared with a microparticulate system, a nanoparticulate delivery system has significantly better absorption and uptake properties in the intestinal lumen and M-cells, respectively. However, Wilkhu et al. noted that the larger-sized liposomes (~6 mm) were more effectively taken up by PPs than the smaller-sized vesicles (2 mm) [48]. This result was inconsistent with Mann et al., who showed that two different populations of larger size vesicles (60–350 and 400–2500 nm) demonstrated higher IgG2a production and antigen-induced IFN-g production compared with smaller vesicles (10–100 nm) [51]. The exact mechanism for a higher immune response from larger size vesicles is still unexplored. Higher immune responses are likely to be correlated with higher antigen-loading capacity in the larger-sized vesicles enabling delivery of a large quantity of antigens for a prolonged period of time. Also, enhanced mucosal penetration due to bile salts, increased lipophilicity of phospholipids and controlled release kinetics of antigens could be possible mechanisms that result in different immune responses. In addition to improved stability in GIT, bilosomes doi: 10.1586/14760584.2014.936852

are stable during freeze-drying and easy to mass produce making them an interesting delivery system. Alternatively, liposomes can be modified as archaeosomes to overcome their low GIT stability. Archaeosomes are liposome vesicles with an anionic archaeobacterial membrane made up of diether or tetraether lipids. Structurally, these anionic lipids are regularly branched, usually with fully saturated phytanyl chains of 20–40 carbon length that are attached to sn-2,3 carbons of the glycerol backbone (s) via ether bonds [52]. The unique membrane-spanning characteristics of archaeal lipids promote better stability to the liposomal vesicles [53]. Archaeosomes are well-tolerated, non-toxic and stable in the GIT and possess longlasting immune-adjuvanting capacity that makes them superior to unmodified liposome in the production of antigen-specific cell-mediated immunity [54–56]. In a mouse study, orally delivered archaeosomes encapsulating model antigen ovalbumin (OVA) induced higher antigen-specific mucosal IgA and systemic IgG responses compared with conventional liposomes [57]. Another major limitation of liposomes is their weak mucouspenetrating capacity. Due to its high degree of lipophilicity, the mucus layer easily clears liposomes and reduces their permeability across the mucosa, which compromises their applicability for oral administration. The highly lipophilic liposome surface can be modified with hydrophilic polymers to reduce its hydrophobicity. Liposomes modified with pluronic F127, a triblock polymer with a central polypropylene glycol arm and two hydrophilic polyethylene glycol arms improved the mucous penetration capacity of liposomes compared with unmodified liposomes [58]. Surface-modified liposomes can be used for selective targeting of M-cells. Lectin-modified liposomes appear promising for the oral delivery of antigens in the animal model. Recently, glucomannan-modified bilosomes encapsulating tetanus toxoid (TT) were used to target the mannose receptor in APCs [59]. After oral administration, these bilosomes showed significantly higher systemic immune responses compared with unconjugated bilosomes, niosomes and alum adsorbed TT. More importantly, a cellular immune response (IL-1 and IFN-g) and a high mucosal-specific IgA response were detected for glucomannan bilosomes in salivary and intestinal secretions. Nanoemulsion

Emulsion-based delivery system is one of the most promising delivery systems and an immunomodulator for vaccine delivery. Nanoemulsion is an isotropic system of two immiscible liquids (oil and water), which stabilize upon addition of an appropriate amount of surfactant to produce nanometer-sized (20–200 nm) droplets [60]. Due to the comparable particle size of nanoemulsions to pathogens, they are easily transcytosed across the intestinal cells. Emulsions are easy to produce at low cost and show potential to solidify (solid emulsions), which could improve vaccine stability during transport/storage and increase the capacity for mass immunizations. Both, water-in-oil- (w/o) and oil-in-water (o/w)-based nanoemulsions have shown potential in encapsulating, protecting and delivering various drugs and Expert Rev. Vaccines



peptides/proteins antigens through the mucosal route [61,62]. The higher immunogenicity of nanoemulsion-based carriers for vaccine delivery is attributed to their ability for passive targeting, increased phagocytosis by APCs, increased blood circulation time and sustained-release capacity [63]. Depending upon the solubility, antigens are either dispersed into aqueous or oil phases to form droplets of w/o or o/w emulsions. Thus, direct contact of antigens with gastric fluids is avoided, which increases gastric stability [64]. Additionally, the presence of surfactant(s) in the emulsion system provides better stability against premature leakage of antigens in the GI fluid, which further enhances their permeation across the luminal wall [60]. Nanoemulsion-based protein antigen formulations were studied as a vaccine against tumors [63,65]. Antigens (a complex of melanoma antigen, heat shock protein and staphylococcal enterotoxin A) incorporated in a nanoemulsion showed high encapsulation efficiency (87%) with a small particle size of 15–25 nm [63]. The antitumor activity of orally administered nanoemulsion showed a similar immune response to the conventional subcutaneous route. In addition, the nanoemulsion showed a higher cellular immune response compared with unencapsulated formulations. Different emulsion-based adjuvants, such as AF03, AS03 and MF59, were considered for vaccine delivery. Among them, MF59 showed an excellent safety profile and has been an approved adjuvant for clinical use in more than 30 countries [66,67]. MF59 is an o/w emulsion-based adjuvant with nano-sized droplets (droplet size ~160 nm), which are formulated with squalane-based oil and stabilized by two non-ionic surfactants (polysorbate 80 and sorbitan trioleate) in an aqueous citrate buffer [66]. Multiple emulsions are also being considered for oral vaccine delivery. These emulsions are a multi-compartmentalized system in which w/o or w/o emulsion droplets are dispersed into external oily or aqueous phase that are further stabilized by external surfactants. The most common forms of multiple emulsions are water in oil in water or oil in water in oil. The main advantages of multiple emulsions are their ability to incorporate both hydrophilic and lipophilic antigens in a single formulation system with comparatively better protection of antigens than in biphasic emulsions. However, the stability of multiple emulsions is difficult to control. On oral administration, OVA encapsulated squalane oil-based multiple emulsion (water in oil in water) formulations were found to be taken up by macrophages and APCs at local mucosal lymph nodes, which induced significantly higher mucosal and systemic antibody titers compared with aqueous solutions [68]. Immunostimulating complexes

ISCOMs were first described in 1984 as a vaccine delivery vehicle [69]. ISCOMs are self-assembling spherical cage-like structured particulate complexes, usually 40 nm in size. They are made up of antigens, cholesterol, phospholipids and Quil A saponins (bark of Quillaia saponaria [QS]) [70]. The ISCOMmatrix, also called ISCOMATRIXTM , is essentially similar in structure to ISCOM but lacks antigens in the complex [71]. informahealthcare.com

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Antigens are usually added in to the ISCOM-matrix at the later stage of the formulation. From an immunological perspective, ISCOMs serve as an antigen carrier due to their particulate nature and adjuvant inclusion (saponin fractions). The leading saponin derivatives that are used in ISCOMS are Quil A and QS 21. Due to its mild toxicity and hemolytic properties, Quil A was limited to veterinary use [69]. The other derivate, QS 21 is a purified saponin and comparatively safer, well-tolerated and has been widely used in vaccine clinical trials against infections, cancer and nervous disorders [72,73]. ISCOMs produce strong humoral as well as cellular immune responses through MHC I and II pathways against a variety of delivered antigens [74,75]. The cellular or protein-based lipophilic antigens are easily entrapped within the lipid complex surface. However, inclusion of hydrophilic antigens in ISCOMs is troublesome and requires modification prior to inclusion. Hydrophilic antigens can be modified chemically by conjugating lipids before inclusion in the ISCOMs, and such modification may have further positive impact on vaccine efficacy [76,77]. ISCOMs are usually used for parenteral delivery. Orally administered ISCOMs, as a vaccine candidate, have shown increased immunogenicity mediated by enhanced recruitment of macrophages and B-cells in the PPs as compared with soluble antigens [78–81]. In addition, ISCOMs has been resistant to bile salts and do not induce tolerance to orally delivered antigens [75]. Mowat et al. incorporated fusion protein (CTA1-DD, a mucosal adjuvant) with OVA323–339 peptide epitope into ISCOMs [80], which showed a high stability in the GIT and no toxic effects. This vaccine was effective even at a low dose of 150 ng per mouse to induce systemic immune responses (IgG2a and IgG1 isotypes). Following oral administration of ISCOM incorporating HSV-2 antigens, high levels of local (IgA) and serum antibody (IgG) were detected [82]. A challenge study with this formulation showed sufficient protection against a heterologous lethal dose of HSV-2. The result suggests that ISCOMs can induce a broad range of immunity. Virus-like particles

VLPs have attracted commercial interest in recent years after the approval of the VLP-based human papiloma virus vaccines, Gardasil (Merck & Co.) and Cervarix (GSK), in the 2000s [83,84]. VLPs are composed of an expressed viral envelope or capsid proteins, which can spontaneously self-assemble. VLPs mimic the 3D conformational structure of a real virus but lack genetic material making them non-infectious [85]. Since VLPs mimic authentic viruses, they are easily recognized by the host immune system. VLPs provide a safer alternative to traditional vaccines based on inactivated or attenuated viruses due to their strong ability to stimulate a strong immune response without the need of external adjuvant, and have lesser side effects, higher stability and easy large-scale production [86,87]. VLPs show a polydispersed, multimodal size distribution similar to the size of intact virions with size ranging from a few nanometers to micrometers [88,89]. VLPs can be engineered at their surface, which makes it possible to insert a repetitive array doi: 10.1586/14760584.2014.936852


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of epitopes (single or multiple) by genetic fusion or chemical coupling. VLPs have shown excellent stability of the attached peptide epitopes against gastric enzymes [90]. VLPs have been studied as oral vaccines against a variety of diseases [91–94]. They possess high affinity for intestinal mucosal surface receptors, which are easily recognized by expressed submucosal APC or intraepithelial lymphocytes. However, not all the VLPs intended for oral administration may express a natural tropism for intestinal epithelial cells. Human immunization with Norwalk VLPs prepared from recombinant capsid protein displayed a significant production of humoral and cellular antibodies [92]. Bovine papilloma virus 1 (BPV) is a non-human pathogen that has been used as delivery platform for antigens through the mucosal route due to its ability to resist GI degradation and its strong immune-activating properties. HIV type 1 gp41 epitope was delivered using the BPV-VLPs platform, which produced epitope-specific systemic IgGs and mucosal IgA antibodies against HIV [93]. Huang et al. demonstrated that BPV-VLP encoded with a truncated carcinoembryonic antigen, expressed by GI cancer cells, showed considerable carcinoembryonic antigen -specific mucosal and systemic cytotoxic T-lymphocyte response [94]. VLPs can also effectively encapsulate DNA and provide sufficient protection of DNA vaccines from degradation. On oral administration, VLPs (prepared from hepatitis E virus) loaded with HIV envelope cDNA sufficiently protected the plasmid DNA and delivered it to intestinal mucosal sites where they elicited mucosal, systemic and cellular immune responses [95]. Despite VLPs being one of the best candidates for commercialization, there are still some concerns regarding their purity. The remaining impurities on VLPs derived from infectious viruses could pose a threat to the safety of vaccines. Moreover, consistent batch-to-batch production of similar sized particles is another hurdle limiting further development of VLPs-based vaccines [96]. Polymeric NPs

Polymeric NPs are defined as nano-sized polymeric particles that have the capacity to encapsulate, adsorb or conjugate any foreign material within itself or on its surface. Over the last two decades, various polymeric platforms for drug and vaccine delivery have been studied [36,97,98]. With the advances in polymer chemistry, different types of natural and synthetic polymers have been used for the oral delivery of antigens. Polymeric NPs have similar advantageous properties as bilosomes. Depending upon the functional characteristics of polymers and the mode of antigen delivery to specific targeted sites, polymeric NPs for mucosal administration are broadly categorized as mucoadhesive NPs, pH-sensitive NPs and specific ligand-attached NPs. The extensively employed biodegradable polymers for the preparation of oral nanoparticulate vaccines are poly lactic acid (PLA), poly lacticco-glycolic acid (PLGA), polyanhydrides and their derivatives. However, NPs prepared by these polymers have certain limitations such as low loading efficiency, hydrophobicity, fast burst release, high manufacturing cost and scale-up difficulty. doi: 10.1586/14760584.2014.936852

In the case of oral delivery, the higher surface area of particles owing to their small size is able to provide increased absorption of NPs across the intestinal epithelium, which results in reduced dosage frequency or volume. Hydrophilic particles are usually transported through normal enterocytes, whereas hydrophobic polymeric NPs are better transported through M-cells [30,99]. Primard et al. demonstrated that negatively charged polymer NPs were effectively taken up by PP within 15 min. The small-sized negatively charged NPs (~200 nm) effectively crossed the mucous layer that led to specific interactions with underlying B-cells and DCs of PPs [100]. Several attempts have been made to enhance the stability of antigens in GI fluids. For example, conjugating polyethylene glycol (PEG) into either side of PLA effectively encapsulated and protected hepatitis B surface antigens in simulated GI fluids and showed high intestinal mucosa uptake [101,102]. Even with a single oral administration, this formulation was able to enhance antigen-specific humoral (IgA and IgG antibodies) and cellular immune responses (Th1) without requiring any booster dose. However, co-polymerization of PEG to PLA reduced the encapsulation efficiency of hepatitis B surface antigens [103]. Garinot et al. used RGD peptides (GRGDS) as the targeting ligand and covalently attached them to the surface of PEGylatedPLGA NPs [30]. The RGD peptides are specific to b1 integrins expressed at the apical side of M-cells. The PEGylated-PLGA NPs protected the antigens (OVA) against GIT degradation and specifically delivered them to the M-cells. It was observed that a significantly reduced antigen dose (5 mg) was sufficient to induce a similar immune response as the high-dose immunization (‡100 mg). In another approach, Sarti et al. used modified lipopolysaccharide, monophosphoryl lipid A as external adjuvant to formulate in PLGA NPs for the oral delivery of OVA antigens. After oral administration, they observed a strong IgG and IgA immune response compared with OVA-PLGA NPs alone [104]. OVA-loaded PLGA particles were actively transported across enterocytes due to their nano-sized particles (320 nm and 20 mV) and the (pseudo) lipophilic nature of PLGA. Furthermore, PLGA NPs slowly hydrolyzed to release OVA antigens and prolonged the antigen presentation to APCs. Recent approaches on oral mucosal delivery of NPs have been directed toward the use of bioadhesive polymers. The mucoadhesive polymers prolong retention time of the particles in the mucus due to interactive forces (steric or adhesive) that provide long-term exposure to the intestinal cells for enhanced absorption. For example, coating of nanospheres with poly (butadiene-maleic anhydride-co-L-DOPA) increased the particle uptake by 10-fold in the small intestine [105]. Conjugating a specific ligand with bioadhesive polymer produced long-lasting mucosal and systemic immune responses. Salman et al. reported that bioadhesive poly(anhydride) NPs coated with mannose or Salmonella enteritidis-derived flagellin with a particle size of 300–400 nm was more immunogenic by the oral route than uncoated ones. In particular, a stronger systemic antibody response (Th1 and Th2 immunity) was observed compared with non-coated particles [106]. Expert Rev. Vaccines



One of the methods to increase the stability of encapsulated antigens is cross-linking of the polymers. In addition, efficiently cross-linked polymer is able to release antigen in a controlled fashion. Poly-dextran aldehyde cross-linked water-soluble polymeric NPs were able to prevent rapid leaking of the antigens. While comparing immunogenicity, cross-linked NPs were shown to induce higher antibody titers than non-cross-linked formulations [107]. To increase the stability of antigens during their transit through the harsh acidic gastric environment to the intestine region, pH-sensitive polymers have been used. These polymers encapsulated, targeted and released antigens in a controlled fashion at the lower region of the intestine [108]. Antigen that can be selectively delivered only to the colon can avoid exposure to the harsh GIT environment and can undergo more efficient transportation across intestinal wall due to the presence of a mild neutral pH, less enzymatic action, low bile salt activity and slow mucosal layer turnover of the colon. However, the presence of a thicker and a denser mucosal layer in the large intestine as compared with small intestine limit the absorption efficiency of particle-based vaccines. Large intestine-based vaccine delivery is mostly influenced by passive enterocytic transportation rather than active PPs-mediated transportation. Recently, a large intestine-targeted oral vaccine was prepared by encapsulating PLGA NPs vaccine inside microparticles (Eudragit FS30D) [109]. Incorporation of NPs (300–500 nm) vaccines in large-sized microparticles sufficiently protected the vaccines from acidic pH and enzymatic degradation. These microparticles were degraded in the large intestine (pH > 7.0) and the NPs were released. After oral administration, significant amounts of antigen specific to IgA and IgG were detected. Challenge studies in mice showed a sufficient specific T-cellmediated response to protect against vaccinia virus vPE-16. A number of studies have demonstrated the use of natural biodegradable polymers such as chitosan and its covalently modified derivates for the oral delivery of protein and DNAbased vaccines against schistosomiasis, breast and ovarian cancer [110–112]. Chitosan and its derivative are mucoadhesive and have the ability to stimulate immune cells either by directly interacting with the M-cells or by opening the tight junctions between the epithelial cells [113]. Positively charged chitosan strongly binds with negatively charged sialic acid (principal component of mucus) by electrostatic interaction and decreases the mucocilliary clearance of encapsulated antigens providing prolonged exposure in the lumen [114]. However, chitosan is only soluble at an acidic pH and lacks good solubility in the intestine and at neutral pH. Thus, its amine moiety was methylated to increase its solubility at neutral pH [113]. Different M-cell-specific targeting ligands were conjugated to chitosan NPs to improve oral delivery of antigens. Yoo et al. chemically conjugated chitosan NPs to M-cell homing peptide (CKS9) as an oral vaccine carrier [112]. These CKS9-conjugated chitosan NPs were able to bind to M-cells in PPs. Alginate is another biodegradable natural polymer used for the oral delivery of vaccine due to its immunostimulatory and informahealthcare.com

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M-cell internalizing properties [110]. Chitosan alone may not provide sufficient protection for antigens due to its immediate burst release of antigens at a gastric acidic pH (1–4). In order to overcome chitosan instability in low pH, cationic chitosan NPs can be coated with gastric acid-resistant alginate. Oliveira et al. found that the electrostatic interaction between the carboxyl group of alginate and the amino group of chitosan formed a polyelectrolyte complex. Chitosan NPs encapsulating antigens were prepared and alginate was adsorbed on the surface of chitosan NPs. These formulation-protected protein antigens, SmRho and legumain-based DNA vaccines, were not degraded in simulated gastric and GI fluids and produced a protective immune response against Schistosoma mansoni infection and breast cancer, respectively [110,111]. Interestingly, the addition of alginate coating in chitosan NPs increased the encapsulation efficiency of antigens from 76 to 95% [110]. Recently, De´moulins et al. showed that alginate-coated chitosan nanogel formulated with Toll-like receptor (TLR) ligands were able to influence DCs endocytosis of the TLR ligands. Nanogel formulated with CpG-ODN (a TLR 9 agonist) induced IFN-a, IL-6 and TNF-a production, whereas with Pam3cysSK4 (a TLR 1/2 agonist) induced IL-1b, demonstrating importance of predefining the optimal combination of immunomodulatory agents and delivery vehicles for optimal vaccine efficacy [115]. In addition, chitosan and alginate are biocompatible and have the capacity for prolonged release of antigens, which makes them an efficient polymeric nanocarrier for the mucosal delivery of vaccines. Inorganic NPs

Inorganic NPs have been investigated as potential oral vaccine carriers due to their attractive physical and chemical properties. The surface morphology and particle size of inorganic NPs can be finely tuned to provide better colloidal stability, high antigen encapsulation efficiency and targeted delivery by attaching ligands [31,116]. The surfaces of inorganic NPs are rigid and stable allowing protection of adsorbed antigens against the harsh GI conditions and preventing premature release of antigens. Gold NPs are inert, biocompatible and their size can be easily tailored in the low nanometer range (1–100 nm) [117,118]. There is growing interest in the use of gold particles for the delivery of vaccines [117,119,120]. The use of such NPs as an antigen delivery platform avoids the production of carrier-directed antibodies [121]. Gold NPs conjugated with different antigenic peptides and polymers (poly acrylic acids and chitosan) are stable in acidic solutions [116,118]. It has been shown that gold NPs conjugated to antigenic peptide, matrix 2 protein, induced an antigen-specific IgG response when administered thorough nasal mucosal routes [122]. They were also efficient for boosting the immune response induced by intramuscularly vaccination of DNA making it suitable as a novel immunoadjuvant [123]. However, a limited number of attempts have been made for oral vaccine delivery using gold-based NPs. In a recent study, Barhate et al. prepared 40 nm, positively charged (+35 mV) chitosan functionalized gold NPs as an oral nanocarrier for the doi: 10.1586/14760584.2014.936852

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delivery of model antigen TT along with immunostimulant QS extract [116]. The developed gold NPs were stable against hydrolysis in GI fluid. In vivo tests showed significant TT-specific IgG and IgA immune responses. Inorganic NPs are a promising vaccine delivery system; however, their role as immune adjuvants in oral delivery is yet to be investigated.


Expert commentary & five-year view

The body’s mucosal system is the most vulnerable site for pathogen infiltration and induction of diseases. Therefore, neutralization of pathogens as they enter the body is more desirable than treatment of pathogens that have gained entry. Among the mucosal vaccine delivery approaches, oral delivery is considered the most preferred route due to several benefits such as high patient compliance, low cost, capacity for mass immunization, requirement for less-trained personnel and avoidance of needle-associated hazards. However, with the exception of liveattenuated or whole killed pathogen-based vaccines, there is no example of a vaccine delivered through the oral route using NPs on the market. One of the reasons for the failure of oral vaccines is antigenic denaturation, especially at the low gastric pH of the stomach (pH < 2). Thus, except for vaccine formulation improvements, feeding of alkalizers to neutralize stomach prior to oral vaccination would be required. The enhanced immunogenicity of NPs could be attributed to their size mimicry of pathogens. However, size is not the only factor for improved immune response. Other factors should be equally considered including surface charge, antigen payload, attached targeting ligands, hydrophobicity, bioadhesiveness and release characteristics. Several mucoadhesive, pH-sensitive and gastricacid resistant biodegradable polymers such as PLGA, PGA, chitosan and alginate alone or in combination have shown significant progress in enhancing the stability of antigen in GI fluid. Lipid-based particulate carriers such as liposomes can be engineered by coating them with different polymers for enhanced protection against degradation and provide a depot effect to prolong the antigenic exposure to APCs. This prior coating can easily produce liposomes in a variety of sizes. Moreover, other shortcomings of delivery systems with low permeability can be improved by specific ligand-mediated targeting to M-cells, epithelial DCs and enterocytes. A combination of the above strategies/factors (rather than a single element) may result in production of vaccines, which can be efficient and safe enough to meet the requirements for human use. A growing issue is the poor in vitro/in vivo correlation when testing the immunogenic efficiency of oral vaccines. Results from in vitro permeation and uptake studies (M-cells and DCs) may not fully correlate to the in vivo situation in mice and the animal model. Although, the majority of immunological studies on oral vaccines are conducted in mice, they are not a perfect model because their population of M-cells is higher than in humans. Thus, the data obtained from mouse experiments might result in misleading conclusions. Animal model for testing human mucosal vaccine candidate should reflect the relevant physiological characteristics closer to human. The doi: 10.1586/14760584.2014.936852

similarities in maturation of the mucosa-associated lymphoid tissues in pig, cow and sheep make a good alternative model for studying the mucosal delivery of vaccines. More importantly, the mucosal immune system is well developed in these animals before birth in contrast to experimental mice, in which mucosal immune system can be still underdeveloped during the experiments [124]. However, the use of large animals for the in vivo study is associated with severe ethics restriction and very high cost. Another, important issue with the NPs-based vaccine is their biological safety. The complete pharmacokinetics and biodegradable profile of nanoparticulate vaccines are little understood. Although most of the synthetic/natural biodegradable polymers are easily cleared by the body, the non-biodegradable polymeric vaccines take a longer time for systemic clearance, which can result in toxic side effects. Therefore, intensive studies on safety profiles of particulate polymers should be performed before translating NP vaccines from laboratory to human use. The currently available human oral vaccines against polio, gastroenteritis, typhoid and cholera are prepared from liveattenuated or whole killed pathogens. Although some of the current research has shown promising results, a licensed oral vaccine based on NPs or microparticles is lacking. Other types of oral vaccines, such as live-attenuated cholera, avian influenza and anthrax vaccines are currently under clinical trials. The main challenge is to transform NPs-based oral vaccines to market. The difficulties of this are related to their complex design, difficult manufacturing process, high cost, lower reproducibility of NPs production process and lack of safer but strong mucosal adjuvants. The recent significant progress in oral NPs vaccine development should finally result in commercial vaccines. The future-generation oral NP vaccines should learn from the various shortcomings associated with the current NP vaccine research and be able to find novel methods for efficient antigen delivery. The improved future oral vaccines will utilize inherent immunoadjuvant properties in the nanocarriers for safe and effective delivery to the GIT. Moreover, it can be expected that highly specific new-generation M-cells, epithelial DCs and enterocytic cells targeting moieties will be explored for antigen delivery to APCs. A novel strategy of conjugating various TLR agonists, such as lipoamino acids and synthetic peptide epitopes, to the biodegradable polymeric particles is expected to overcome stability and permeability issues. Alternatively, glycosylation of the synthetic antigenic peptides might be used to improve hydrolytic stability and intestinal membrane permeability. It can be assumed with reasonable certainty that in the not so far future NP-based vaccines will overcome the barriers presented by GIT and will selectively target intestinal immune cells without toxic side effects. The only question remaining is if they can be produced by economically feasible processes to allow low-cost mass vaccination as oral immunization has proven, especially advantageous in the world’s developing areas. Financial & competing interests disclosure

This work was supported by National Health and Medical Research Council (NHMRC), Australia. The contents are solely the responsibility of Expert Rev. Vaccines


authors and do not necessarily represent the official views of NHMRC. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial

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conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues • The main challenges associated with oral vaccine delivery are sufficient protection of the integrity of the antigens and effective transportation across the intestinal epithelium. • Efficacy of oral vaccines is mostly hampered by the low population of microfold-cells (M-cells) in the intestines. • An alternative novel targeted delivery system is sought for effective delivery of oral vaccines to M-cells, intestinal dendritic cells and enterocytes. Expert Review of Vaccines Downloaded from informahealthcare.com by Tulane University on 09/30/14 For personal use only.

• Several polymer and lipid-based nanocarriers have shown potential to enhance the immunogenicity of oral vaccines; however, there is no clear specific mechanism identified to show how these carriers promote enhanced mucosal and immune responses. • Certain modifications of existing nanoparticulate delivery systems may drastically change stability/efficacy of vaccines. For example, bilosomes in contrast to liposomes have shown greatly improved stability in the gastrointestinal tract and during production/storage. • Further developments in nanoparticle vaccines should concentrate on simple and robust design of the formulations for easy scale-up and commercialization.

ex vivo measurement of vaccine-induced human humoral immune responses in blood. Nat Protoc 2013;8(6):1073-87

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Mucosal immunity to oral vaccines.

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Oral adjuvants for viral vaccines in humans.

Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

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