Recent advances in oral vaccine development

Abstract

Oral vaccination is the most challenging vaccination method due to the administration route. However, oral vaccination has socio-economic benefits and provides the possibility of stimulating both humoral and cellular immune responses at systemic and mucosal sites. Despite the advantages of oral vaccination, only a limited number of oral vaccines are currently approved for human use. During the last decade, extensive research regarding antigen-based oral vaccination methods have improved immunogenicity and induced desired immunological outcomes. Nevertheless, several factors such as the harsh gastro-intestinal environment and oral tolerance impede the clinical application of oral delivery systems. To date, human clinical trials investigating the efficacy of these systems are still lacking. This review addresses the rationale and key biological and physicochemical aspects of oral vaccine design and highlights the use of yeast-derived β-glucan microparticles as an oral vaccine delivery platform.

Keywords: :

• GALT
• Peyer’s patches
• antigen delivery vehicles
• microparticles
• oral
• vaccination
• yeast-derived beta-glucan

Introduction

The mucosal epithelium is the major entry site for oral pathogens, which creates the need for proper induction of local immune response to prevent infectious disease. Diarrheal disease is the second leading cause of death in children under 5-y-old, killing around 760 000 children annually.¹ Both respiratory and gastro-intestinal infections kill approximately 5 million children under the age of 5 in developing countries and cause more than 10 billion disease episodes annually.² Oral vaccination appears to be the only route that protects against enteric pathogens. If oral vaccination were used to prophylactically treat susceptible human populations at risk of enteric pathogens, it could prove to be the most cost-effective and efficient manner to reduce morbidity and mortality against gastro-intestinal infections.^3,4 Successful oral vaccination will extend effective immunity to the small intestine, ascending colon, salivary and mammary glands, due to the homing of activated antigen-specific lymphocytes to remote mucosal effector sites.⁵

Currently, most oral vaccines are based on heat-killed or attenuated pathogens. The downside to these whole pathogen-based vaccines is that they do not provoke a sufficient immune response, which consequently results in pathogen persistence and disease progression in immunocompromised individuals. For example, in 2000, outbreaks of poliomyelitis were reported in Egypt, Haiti, the Philippines, and the Dominican Republic.^6,7 These outbreaks of polio were caused by live attenuated polio vaccine (OPV) that was orally administered. The cause of this polio outbreak was due to a reversion of the attenuated virus back to the infectious wild type strain resulting in increased neurovirulence and thus leading to infections among immunocompromised individuals.⁸ A safer alternative might be a protein subunit vaccine, which is composed of specific antigens found within the pathogen. However, soluble antigens do not penetrate the mucosal lining of the intestine and are consequently less efficiently presented by antigen presenting cells (APCs).⁹ These problems prevent an efficient mucosal and/or systemic immune response following oral vaccination and inhibit the ability to successfully develop new oral-based vaccines.

Undoubtedly, vaccination constitutes one of the major breakthroughs in human medicine, which has allowed the eradication of numerous infectious diseases.¹⁰ Oral vaccination provides both social and economic advantages, especially in developing countries. The use of needle-free vaccine administration eliminates the risk of transmitting blood-borne pathogens and can be performed by health workers without any medical training. Furthermore, oral vaccination increases patient compliance because the pain and discomfort from a needle-stick is avoided. This ultimately results in an increased adherence to vaccination schemes. In addition, since the gut is already heavily colonized by microbiota, oral vaccine formulations do not require extensive antigen purification, simplifying the overall manufacturing process. Taken together, the simplified production, storage, and administration method for oral vaccination might preferentially favor it over conventional needle methods during pandemic situations.¹¹

Biodegradable and biocompatible microparticulate antigen carriers are novel and promising vaccination methods that may act synergistically as a delivery vehicle and an adjuvant to ensure the proper induction of both cellular and humoral immune responses.^12,13 Microparticulate encapsulation strategies should ideally protect the antigen from degradation and increase the concentration of antigen in the vicinity of mucosal tissue for better absorption. Additionally, microparticulate antigen carriers can selectively target the gut-associated lymphoid tissue (GALT), Peyer’s patches (PP) and intestinal APCs to initiate immune responses.¹⁴ The categories of particulate antigen delivery systems, including immune stimulating complexes (ISCOMs), liposomes, virus-like particles, micro- and nanoparticles have been widely reviewed in recent literature.^15-18 In this review, we discuss the challenges regarding the development and design of oral vaccine formulations, focusing on the documented effects of yeast-derived β-glucan microparticles as an oral delivery platform.

Challenges in the Development of Oral Vaccine Formulations

The oral delivery route is the most challenging route in mucosal vaccination and many factors influence the uptake of particulate antigen carriers by the intestinal epithelium. The physiology of the gastro-intestinal tract, which is reinforced with various defense and clearance mechanisms, contributes to the equilibrium between immunity and oral tolerance in the gut. Gastric antigen degradation can be circumvented by using pH-sensitive polymers, (e.g., methacrylic acid,¹⁹ hydroxypropyl methylcellulose phthalate²⁰) that protect antigens at acidic pH and respond to the slightly basic pH in the intestine due to their intrinsic ionizable functional groups leading to swelling or dissolution of the vehicle and site-specific antigen release. In contrast, other types of pH-sensitive polymers such as polymersomes dissolve under acidic conditions, which are particularly suitable for targeting tumors and endolysomoses.^21,22 Currently, enteric coating based on Eudragit (poly (methacrylic acid-co-ethylacrylate) copolymers) formulations, which remain impermeable at the low gastric pH and dissolve upon alkaline pH in the small intestine, are extensively employed.²³ Combinations of Eudragit L100–55 or S-100 with other polymers have shown to improve the oral bioavailability and the controlled release of drugs.^24-27 Shastri et al.²⁸ reported enhanced antigen-specific IgG1 and IgG2a antibodies and protection against live influenza viral challenge after 2 oral immunizations with microparticles containing inactivated influenza A/PR/34/8 H1N1 virus with Eudragit S100 and trehalose.

In addition to the wide pH gradients in the gastro-intestinal tract, the antigen is subjected to different mechanical features such as the intestinal peristaltic movement, capture by mucus and glycocalyx and attack by digestive enzymes (proteases, lipases, nucleases, bile, lactoferrin, and peroxidases), which results in antigen dilution and degradation. In this regard, an innovative approach based on particle surface modification with bio-adhesive molecules like plant lectins, microbial adhesins, specific antibodies, or Toll-Like Receptor (TLR) ligands may target the epithelial barrier through carbohydrate or receptor binding. Lectins, such as Aleuria aurantia lectin, wheat germ agglutinin or Ulex europaeus agglutinin-1 (UEA-1), are promising targeting moieties that result in M-cell-specific adherence. Several studies have shown that UEA-1-coated particles selectively bind to M-cells in murine PP upon oral gavage or injection into ligated intestinal loops.^29-31 In humans, few M-cell markers have been reported, cathepsin E,³² sialyl Lewis A antigen³³ and clusterin³⁴ however, their real potential as human M-cell targeting molecule awaits further investigation. An area gaining more importance is targeting vaccines to DCs together with TLR agonists, for instance, TLR9 ligands or CpG-containing oligodeoxynucleotides (CpG-ODNs). A study by Hunter et al.³⁵ showed that oral/nasal/vaginal immunization with PLG microparticles containing group B Streptococcus antigen and CpG-ODN, elicited both IgG and secretory IgA antibodies in blood and vaginal washes in mice. Multiple studies demonstrate that soluble antigen mixed with free CpG failed to induce a potent antigen-specific immune response. This contrasts with PLG particles containing CpG absorbed onto the particle surface or PLG particles containing CpG co-encapsulated with antigen inside of particles, which generate potent immune responses.^36,37

Antigen transport is restricted by epithelial barriers created by tight cellular junctions. Smith et al.³⁸ showed that the mucoadhesive molecule chitosan reorganized the structure of the tight junction proteins occludin and zonula occludin protein-1 in Caco-2 cells, enhancing its permeation and prolonging its residence time. However, chitosan is insoluble in water at physiological pH, but its quaternized derivative N-trimethyl-chitosan (TMC) exerts high solubility over a wide pH range, which makes it favorable. In addition, TMC possesses intrinsic adjuvanticity.^39-41

Another major challenge in oral vaccine development is oral tolerance, which downregulates cell-mediated and humoral immune responses in the gastro-intestinal tract to ensure local homeostasis. As the mucosal absorption of soluble antigens is generally very low, preferential induction of tolerance occurs in the absence of adjuvants. However, particulate antigens are more effective at stimulating protective immunity and cross-presentation due to their adjuvant properties discussed later. Many particulate vaccine strategies including β-glucan microparticles,⁴² PLGA,⁴³ exosomes, virus-like particles⁴⁴ and spray-dried polyelectrolyte microspheres⁴⁵ have been reported to exert efficient cross-presentation at up to 1000-fold lower antigen doses. It has also been reported by De Koker et al.⁴⁶ that OVA-loaded polyelectrolyte capsules strongly enhance antigen presentation to both CD4⁺ OT-I and CD8⁺ OT-II cells, allowing cross-presentation at a 50-fold lower antigen dose when compared with soluble OVA alone.

A second group of factors affecting antigen processing and efficiency of antigen uptake are associated with the host itself such as species, age, genetic nature, and nutritional and health status. Oral vaccines against rotavirus, V. cholerae and Escherichia coli (ETEC) have been found to be less effective in developing countries, ascribed to the differences in nutritional status, natural/maternal antibodies, poor sanitation, and gut microflora like ongoing persistent infections with helminths and parasites.⁴⁷ Nutrition-related factors such as malnutrition, vitamin A or zinc deficiency, and malabsorption of nutrients as a consequence of intestinal inflammation, may all result in diminished mucosal immunity in response to oral vaccination.^48,49 Data with regard to the impact of vitamin A deficiency on the immunogenicity and protective vaccine efficacy are conflicting. Although most studies could not demonstrate an effect of vitamin A deficiency or supplementation on antibody levels in response to vaccination,⁵⁰ some clinical and animal studies showed diminished antibody responses.^51,52 Jaensson-Gyllenback et al.⁵³ described that vitamin A deficiency directly impairs intestinal CD103⁺ dendritic cell function. Retinoic acid (RA), a vitamin A metabolite, has been shown to be a crucial regulator in intestinal immunity and homeostasis by promoting IgA class switch recombination, plasma cell differentiation, and development of regulatory T-cells.⁵⁴ Furthermore, antibodies from breast milk or the maternal placenta may also impede mucosal vaccine immunogenicity.

Finally, the factors associated with physicochemical properties of antigen delivery vehicles, such as surface charge, size, hydrophobicity/hydrophilicity balance, antigen dose, and total number of particles per dose, all need to be considered in the design of safe and efficacious oral vaccines.

Vaccine Design

The development of an effective oral vaccine delivery system requires careful consideration of its physicochemical properties. Ideally, these systems should (1) be safe, (2) overcome the harsh gastro-intestinal environment, (3) target the immune inductive sites, and (4) induce long lasting immunological memory. Particle size is of utmost importance for the in vivo distribution, fate, and targeting ability of the oral carrier. It is generally known that nanoparticles are better absorbed than microparticles.⁵⁵ For instance, according to Desai et al.,⁵⁶ 100 nm nanoparticles in rats were 15- to 250-fold better absorbed than 500 nm and 10 µm particles, respectively. Nevertheless, many reports describe that both APCs and PP M-cells are able to internalize particles up to 10 µm through phagocytosis or macropinocytosis.^57-60 Indeed, particulate vaccines typically within the 0.1–10 μm range, resemble the dimensions of common bacteria and viruses, and therefore are recognized by the immune system and readily phagocytosed by APCs. Moreover, small particle internalization occurs through receptor-mediated endocytosis in the PP. Small particles are subsequently disseminated to the mesenteric lymph nodes, blood circulation, and spleen, whereas larger particles are retained in the lymphoid tissue.^61,62 In addition, particle size also plays a role in antigen loading, antigen release, and particulate carrier stability. Nano- and submicron-sized particles exhibit a large surface area, which accelerates their carrier dissolution and antigen release. In contrast, larger particles allow higher antigen encapsulation and slower antigen release.

Surface hydrophobicity and surface charge are likewise contributing factors in the efficiency of particle uptake in the gastro-intestinal tract. More hydrophobic and polymeric particles, e.g., polystyrene and PLGA particles show a strong affinity to PP M-cells due to increased permeability through mucins and appear to recruit phagocytic cells more efficiently.^63,64 On the other hand, hydrophilic particles like cellulose and cellulose acetate are preferably translocated to intracellular compartments. Although positively charged particles interact more efficiently with the negatively charged mucins and cell membranes, Shakweh and Jung reported that negatively charged particles and neutral particles exhibited a greater affinity to PP over positively charged nanoparticles.^57,65 In turn, Nakanishi et al.⁶⁶ demonstrated that positively charged particles induced a more efficient cytotoxic T-cell response in mice. Other advantages of particulate delivery systems are the possibility for high antigen encapsulation, depot formation, and co-delivery of antigens and adjuvants.

Beta-Glucans

Beta-glucans are carbohydrate polymers found in the cell walls of fungi, yeast, plants and bacteria, but also in cereals like oat and barley. Depending on the source, β-glucans contain primary structural variations and physicochemical parameters (solubility, primary structure, molecular weight, and branching), which may influence their physiological functions. Βeta-glucans have been shown to elicit a broad range of functions.⁶⁷ First of all, β-glucans are known to be potent activators of the innate immune system. Several in vitro studies have demonstrated the intrinsic adjuvant capacity of β-1,3-D-glucans from yeast and fungi in enhancing the functional activity of neutrophils, macrophages, DCs, and epithelial cells. For instance, Saccharomyces cerevisiae glucan increased TNF-α production in rat alveolar macrophages in vitro.⁶⁸ Incubation of human whole blood with PGG-glucan (a soluble β-1,3-glucan) enhanced the oxidative burst by leukocytes and increased their antimicrobial activity, all in the absence of inflammatory cytokine production.⁶⁹ Second, β-1,3-D-glucans exert beneficial effects on T- and B-cell responses. Beta-glucan binding to dectin-1 on DCs and macrophages triggers their activation and maturation and enhances the production of pro-inflammatory cytokines such as TNF-α, IL-6, IL-2, IL-10, and IL-23. The aforementioned cytokines are able to prime CD4⁺ T-cells and instruct their differentiation into Th1 and Th17 T-helper cells.⁷⁰ Additionally, dectin-1-mediated recognition of β-glucan induces the activation of antigen-specific CD8⁺ cytotoxic T-lymphocytes (CTL).⁷¹ Third, β-1,3-D-glucans possess anti-infective activity (stimulates innate immune responses) against various bacterial, viral, protozoan, and fungal diseases. Beta-glucans may act as an immunotherapeutic agent in the treatment of a number of diseases. For instance, lentinin (from shiitake mushrooms) enhanced host resistance against Mycobacterium tuberculosis and Streptococcus pneumoniae in mice and oat β-glucan had potent activity against HIV-1 in human peripheral blood mononuclear cells.^72-74 The Ostroff group showed that orally administered yeast β-glucan particles increased survival time in mice challenged with the anthrax bacteria.⁷⁵ Finally, it has been shown that β-glucans have immunomodulatory and cytotoxic effects in cancer by inhibiting tumor growth and increasing survival. Supplementing chemotherapy treatment with β-glucans from Grifola frondosa-extracted Maitake D-Fraction resulted in decreased lung, liver, and breast tumor sizes in 60% of the patients as compared with chemotherapy alone.⁷⁶ Similarly, combining Schizophyllan (derived from Schizophyllan commune) with conventional chemotherapy improved the long-term survival rate in patients with ovarian cancer.⁷⁷

Yeast-Derived β-Glucan Microparticles as a Delivery Platform

The β-glucan particle (GP) delivery platform features efficient antigen loading and receptor-targeted uptake in APCs combined with the inherent adjuvant function. GPs are highly purified, hollow, porous 3–4 micron cell wall shells (Fig. 1) manufactured by treating baker's yeast S. cerevisiae with a series of alkaline, acid, and solvent extractions. S. cerevisiae glucan consists of β-1,3-linked glycopyranosyl residues with smaller numbers of β-1,6-linked branches (Fig. 2). After alkali and acidic treatment, these hollow yeast shells are composed primarily of β-1,3-glucan (>85%) chitin/chitosan (2%) and are devoid of proteins, lipids (1%), and mannans.⁷⁸

Figure 1. Transmission electron micrograph (A) and scanning electron microscopy (B) images of ovalbumin-loaded β-glucan particles (De Smet et al.⁶⁰).

Figure 2. Structure of yeast β-glucan (adapted from Volman et al.¹¹²). Polymer of β-(1–3)-D-glycopyranosyl units with branching at β-(1–6)-D-glycopyranosyl units. S. cerevisiae structure consists of β-1–3 and small numbers of β-1–6 branches and β-1–6 linkages.

CR3 and dectin-1 are 2 membrane β-1,3-glucan receptors that have been characterized at the molecular level. GPs are predominantly recognized by dectin-1 (also known as CLEC7A), a C-type lectin receptor that is highly expressed on cells of myeloid origin, including DCs, macrophages, monocytes, and neutrophils.^79,80 The second receptor is the iC3b-complement receptor 3 (CR3, also known as Mac-1, CD11b/CD18, or α_Mβ₂ integrin), which is highly expressed on neutrophils, monocytes, macrophages, and natural killer cells.^81,82 CR3 is involved in the anti-tumor properties of β-glucans as studies by Xia et al.^82,83 have shown that β-glucan-CR3-mediated binding triggered cytotoxic degranulation on iC3b-coated tumor cells. In addition to dectin-1 and CR3, other receptors have been reported to bind β-glucans, including lactosylceramide, scavenger receptors, and CD5.^84,85 GPs are also potent activators of the alternative complement pathway, which leads to deposition of opsonic C3 fragments on the surface of GPs. GPs stimulate DCs to produce cytokines such as IFN-γ and IL-17, which are associated with beneficial responses in vaccine models of pathogen protection. Moreover, synergistic DC cytokine responses are seen when GPs are combined with TLR agonists.

Microparticulate β-Glucans Stimulate Innate Immunity

A number of groups studied the innate immunostimulatory activity of both soluble and particulate β-glucan formulations from S. cerevisiae.^86-90 The group of Hunter gave mice daily oral doses of 0.1 mg kg⁻¹ of microparticulate β-glucan preparations for 2 wk, which significantly increased the phagocytic activity of mouse peritoneal macrophages.⁸⁷ In-depth in vitrostudies of β-glucan microparticulate-treated peritoneal macrophages by Hunter et al.⁸⁸ showed enhanced B7.1 and B7.2 co-stimulatory molecule expression, which are mandatory as a co-signal in T-cell activation and proliferation. Berner et al.^89,90 demonstrated that β-glucan microparticulates are rapidly phagocytized by peritoneal macrophages and induced the upregulation and secretion of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β. This response was even more enhanced upon IFN-γ priming. Previous reports by Hoffman et al.⁹¹ and by Abel and Czop⁹² described similar cytokine profiles in rat alveolar macrophages and human monocytes. In accordance, a recent study by Huang et al.⁹³ described likewise a strong TNF-α production by IFN-γ primed human DCs upon yeast glucan particle stimulation. This striking increase of cytokine production is caused by IFN-γ priming, which is believed to lower the response threshold of innate cells to microparticulate β-glucans.

Use of Innate Immunity Against Cancer and Infectious Disease Challenge

Several groups made beneficial use of the innate immunomodulatory activity of these β-glucan formulations to protect against cancer and infectious disease challenge. Kournikakis et al.⁷⁵ reported that subcutaneous injection or oral administration of yeast β-glucan particles prior to anthrax challenge in mice, increased the survival rate, reduced pulmonary bacterial load and increased the proportion of bacteria-free animals.

Hurtgen and colleagues performed an intranasal challenge with Coccidioides spores in mice, after the subcutaneous vaccination with yeast glucan particles loaded with an epitope-based vaccine.⁹⁴ Vaccinated mice exhibited an adequate protection against Coccidioidomycosis as featured by lung infiltration of activated Th1, Th2, and Th17 cells, significant reduction of colony forming units and increase of survival compared with non-vaccinated mice.

Two studies by Hong elaborate on the impact of the delivery of whole β-glucan particles in different murine tumor models.^95,96 Orally delivered β-glucan particles are preferentially internalized by macrophages, which migrate to the spleen, lymph nodes, and bone marrow.

Upon processing, small fragments of β-glucan particles bind to the CR3-receptor of granulocytes that trigger cytotoxicity of iC3b-opsonized tumor cells. The combined treatment of β-glucans and antitumor antibodies induced significant tumor regression. Also Li et al.⁹⁷ evidenced reduced tumor burden upon oral particulate β-glucan administration in mice and CR3-dependent neutrophil induced cytotoxicity. In addition, β-glucan particles interacted with DCs and augmented antitumor CD4⁺ and CD8⁺ T-cell responses.

β-glucan Particles as Orally Delivery of Nucleic Acids (siRNA, DNA)

Importantly, GPs have been effectively used for both oral- and parenteral-targeted delivery of a wide range of drug classes (small molecules, siRNA/DNA, peptide/protein antigens, and nanoparticles) in preclinical animal models.^93,94,98-103

Based on the in situ layer-by-layer synthesis through electrostatic interactions, the group of Soto initially engineered yeast cell wall particles for DNA delivery.¹⁰⁴ This encapsulation strategy enables a high nucleic acid capacity and delivery, in addition to the oral bioavailability of β-glucan particles and their inherent β-glucan receptor targeting capacity toward APCs. However, a disadvantage associated with the sequential adsorption of polymers in particular of polyethylenimine (PEI) is that high concentration ranges are cytotoxic. Huang et al.¹⁰² eliminated this PEI-related toxicity, by using alternating hydration and lyophilization steps in the loading process of antigen inside the GP shells. Nonetheless, using this nanomaterial engineering approach, the GP delivery technology platform has been expanded for co-formulating TLR adjuvants and/or siRNAs in discrete layers around the antigen core providing for multiplexed co-delivery to augment vaccine efficacy and safety.

The group of Aouadi designed β-1,3-D-glucan-loaded siRNA particles for oral targeting of M-cells and oral siRNA delivery to mouse macrophages.¹⁰⁵ Oral gavage with glucan-encapsulated siRNA particles once daily for 8 consecutive days, resulted in internalization by GALT macrophages, in which siRNA-mediated gene silencing occurred and subsequently migrated to spleen, liver, and lung. Also Tesz and colleagues combined this β-glucan receptor targeting and high RNA encapsulation capacity of glucan shells to obtain gene silencing in phagocytic cells, following intraperitoneal administration.¹⁰⁶

Microparticulate β-Glucan Sampling Across the Intestinal Mucosae

Besides their application as DNA delivery system, the use of β-glucan microparticles as oral vaccine delivery platform has recently gained increased interest in the pharmaco-medical field. Figure 3 introduces the pathways of β-glucan microparticle internalization in the GALT, in which M-cells, epithelial cells, and dendritic cells are suggested as pivotal players in translocation across the intestinal epithelium.

Figure 3. Schematic representation of the pathways of β-glucan microparticle uptake. Beta-glucan microparticulate antigen uptake in mucosal inductive sites may occur through 3 different pathways: M-cells, intestinal epithelial cells, and dendritic cells. (Route 1) Non-migratory gut-resident CX₃CR1-expressing dendritic cells protrude dendrites through epithelial tight junctions into the lumen to directly sample luminal antigen. (Route 2) Transcellular uptake of β-glucan microparticles across enterocytes by endocytosis. (Route 3) M-cells are specialized in antigen uptake because of their sparse glycocalyx, limited microvillus border, reduced enzymatic activity, and active transcytotic pathway. Transcytosis is a particular process by which M-cells endocytose antigens at their apical membrane, resulting in antigen transport into endosomal tubules, vesicles and large multivesicular bodies and finally exocytosis into the basolateral pocket. FAE, follicle-associated epithelium; SED, subepithelial dome; IFR, interfollicular region; HEV, high endothelial venule.

A variety of in vitro and in vivo models were used to study the interaction between β-glucan-based carrier systems and the intestinal epithelium and are predominantly focused on M-cells. Ahmad et al.¹⁰⁷ addressed the uptake and translocation of yeast-derived glucan microparticles across an in vitro M-cell-like model of human intestinal PP, based on the co-culture of Caco-2 cells and Raji B lymphocytes. Surface binding and transport of yeast-derived glucan particles occurred more efficiently across co-cultures vs. mono-cultures. In contrast, β-glucan microparticles (GP) were efficiently and rapidly internalized by Caco-2 and HT-29 cells, modeling the human small and large intestinal epithelial cells respectively. Cellular uptake of GP increased with incubation time and was dose-dependent.⁶⁰

In 1998, Beier and Gebert investigated the kinetics of S. cerevisiae in PP of minipigs by means of intestinal loops. Yeast particle uptake in the gut of minipigs takes about 1 h of transport from the gut to the underlying immune cells.¹⁰⁸ After 4 h, the majority of the yeast cells were present in phagocytes populating the subepithelial lymphoid tissue. A 24 h incubation period yielded no detectable yeast particles in the PP domes, which is suggestive of biodegradation by phagocytes that migrated to the mesenterial lymph nodes. TEM imaging of murine PP following GP inoculation for 1 h in intestinal ligated loops by De Smet et al.⁶⁰ confirmed endocytosis and transport of GP by M-cells occurred through transcellular pathways. Overall, these studies suggest that M-cell internalization and transcytosis of β-glucan particles are rapidly advancing processes and therefore the most principal gateways in PP.

Oral Immunization with Microparticulate β-Glucans

Another focus is the impact of oral vaccination with antigens and β-glucans as antigen delivery platform to promote enhanced adaptive immune responses. Two groups investigated oral immunization with β-glucan microparticles carrying model antigens such as OVA and BSA. A study by Berner demonstrated that microparticulate β-glucan conjugated to BSA was able to initiate an enhanced IgG response against BSA in both intradermal and oral immunizations.⁹⁰ Food pellets coated with MG/F-BSA conjugates were orally administered to mice as primary immunization and booster doses after 24 d. This MG/F-BSA conjugate induced a significant adaptive immune response, characterized by elevated IgG titers on day 29 and up to a 100-fold increase on day 35 in comparison with other groups.

De Smet et al.⁶⁰ investigated the effect of oral microparticulate OVA-loaded glucan particles (GP-OVA) on local and systemic humoral responses in mice. Animals were sensitized for 3 consecutive days with either 100 µg or 300 µg OVA-containing formulations and boosted on days 14 and 28. Oral administration of GP-OVA induced increased OVA-specific IgA, secretory-IgA and secretory component production in intestinal fluids. Moreover, adoptive transfer experiments in OT-II transgenic mice showed proliferating OVA-specific CD4⁺ T-cells mainly in the spleens of GP-OVA-fed mice. This OT-II proliferation is accompanied by an increased IL-17 production and a tendency toward increased levels of IFN-γ, characteristic for a mixed Th17/Th1 response. The most crucial cellular players, antibody and T-cell mediated responses upon β-glucan microparticulate internalization are depicted in Figure 4. This is in agreement with a study performed by Huang et al.¹⁰⁹ wherein subcutaneous administrations of GP formulations elicited strong antigen-specific antibody and T-cell responses, including the production of IFN-γ and IL17a by CD4⁺ Th1 and Th17 cells.

Figure 4. (A) Activated dendritic cells may migrate to the mesenteric lymph nodes and process the β-glucan particles into peptides that are presented to T-cells. Presentation of β-glucan microparticulate antigen by dendritic cells via MHC class II molecules to CD4⁺ helper T-cells, leads to the activation, proliferation, and differentiation of antigen-specific Th17 and Th1 cells. Furthermore, dendritic cells and the intestinal epithelium produces cytokines such as BAFF, APRIL, and TGF-β1 that trigger the process of isotype switching and differentiation of IgA-committed B cells to IgA-producing plasma cells, which produce dimeric IgA. (B) Mature APCs and antigen-primed lymphoid cells will travel along the mesenteric lymph nodes, through the thoracic duct into the blood stream and end up at mucosal effector sites such as epithelia and lamina propria, where a cellular (effector T-lymphocytes) and humoral (secretory IgA-production by plasma cells) immune response is generated. CCR9 or CCR10 is expressed on gut-homing B-cells and T-cells which interact with CCL25 or CCL28 on the epithelium of the small or large intestine, respectively. IgA, immunoglobulin A; SC, secretory component; S-IgA, secretory IgA.

Conclusions and Perspectives

Contemporary developments in the use of delivery systems and adjuvants have undeniably improved the effectiveness of oral vaccination in animal models. However, knowledge obtained from animal models has not been extrapolated to human beings. The use of particulate systems to deliver clinical relevant antigens has yet to be established in humans. In particular, the differences in microbiome, physiology, immunogenicity, and genetic diversity between animals and humans, may affect vaccine safety and effectiveness. To address the gaps in our understanding of the human mucosal immune system, a full investigation of the unique properties of mucosal surfaces (e.g., gastrointestinal tract) are indispensable. Currently, all licensed oral vaccines are composed of attenuated or inactivated forms of disease-causing pathogens. However, these types of vaccines have been shown to have safety concerns as discussed earlier for polio. In addition, soluble antigens do not sufficiently penetrate the mucus layer of the intestine, are taken up less by APCs and will rather generate tolerance since the host strives to maintain mucosal homeostasis by responding to mucosal antigens with tolerance. For these reasons, currently no subunit vaccines are listed among those approved for oral vaccination.

An area requiring additional effort and consideration is the identification, exploration, and use of targeting molecules such as lectins, antibodies, and TLR ligands to target M-cells and DCs, or immune-inductive sites (e.g., Peyer’s patches). Surface modification of particulate carriers with targeting molecules may enhance carrier uptake and promote optimal immune activation without the induction of adverse reactions. A deeper understanding of ligand-receptor interactions and an in-depth investigation of both the biological and technological aspects relating to these vaccine tailored formulations may contribute to the translational research into humans.

Furthermore, the pharmaceutical industry is charged with many constraints on costs, formulation stability, shelf-life, chemical and physical processes that might cause antigen damage and cold-chain-constrained settings regarding oral vaccine design. Newer generation oral vaccines must comply with high safety, stability, and immunogenicity standards and must generate protective and therapeutic immune responses.

Particulate β-glucans may serve as a suitable vaccine delivery platform because they are FDA approved as GRAS (generally recognized as safe) since 2007 and established in large animals and humans. In addition to their cost-effectiveness in large-scale manufacturing, they exert intrinsic adjuvanticity and are potent activators of the innate immune system suitable for both immunocompetent and immunocompromised subjects.^110,111 As vaccine delivery platform, β-glucan microparticles have a large antigen payload and antigen sparing capacity with the opportunity of co-delivery of both antigen and secondary adjuvant. Their β-1,3-D-glucan composition along with their 3–4 micron size enables receptor-targeted uptake by M-cells and APCs. However, several issues concerning the switch from liquid to dry formulations, the use of a relevant cancer or infectious disease antigen that provides protection in a mammalian system, still awaits further investigation.

In conclusion, the impact of particle characteristics on the human intestinal epithelium and lymphoid tissue along with the role of cytokines in the development of immunity and tolerance still needs to be elucidated. Therefore, a tight network between preclinical research and clinical vaccine development may provide new vaccine options for both human and animal health worldwide.

Abbreviations:
DC	=	dendritic cell
APC	=	antigen presenting cell
GALT	=	gut-associated lymphoid tissue
PP	=	Peyer’s patches
M-cell	=	microfold cell
OVA	=	ovalbumin
GP	=	beta-glucan particle
TLR	=	Toll-like receptor

Acknowledgments

R.D.S. is supported by a doctoral grant from the Concerted Research Action of the University of Ghent (BOF10/GOA/21, project number: 01GC2110W; Ghent, Belgium). L.A. is supported by a doctoral grant from the Special Research Fund of Ghent University (01D41012).

10.4161/hv.28166