Chemical Synthesis, Versatile Structures and Functions of Tailorable

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Chemical Synthesis, Versatile Structures and Functions of Tailorable Adjuvants for Optimizing Oral Vaccination Lei Zhang, Chaohua Hu, Wendi Yang, Xiaolin Liu, and Yunkun Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10470 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016

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ACS Applied Materials & Interfaces

Chemical

Synthesis,

Versatile

Structures

and

Functions of Tailorable Adjuvants for Optimizing Oral Vaccination

Lei Zhang,† Chaohua Hu,‡ Wendi Yang,† Xiaolin Liu,† Yunkun Wu* † †

State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of

Matter, Chinese Academy of Sciences, Fuzhou 350002, China. ‡

National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry

University, Fuzhou 350002, China

ABSTRACT: Oral vaccines have become a recent focus because of their potential significance in disease prevention and therapy. In the development of oral vaccine-based therapeutics, synthetic materials with tailorable structures and versatile functions can act as antigen conveyers with adjuvant effects, reduce the time cost for vaccine optimization, and provide high security and enhanced immunity. This review presents an overview of the current status of tailoring synthetic adjuvants for oral vaccination, modification strategies for producing effectors with specific

ACS Paragon Plus Environment

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structures and functions, enhancement of immune-associated efficiencies, including the barriercrossing capability to protect antigens in the GI tract, coordination of the antigens penetrating mucosa and cell barriers, targeting of concentrated antigens to immune-associated cells, and direct stimulation of immune cells. Finally, we focus on prospective synthetic adjuvants that facilitate the use of oral vaccines via two approaches, in vivo antigen expression and cancer immunotherapy.

KEYWORDS: Immune protection, Oral vaccination, Synthetic adjuvant, Tailorable structures, Antigen delivery, Immune enhancer.


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1.

Introduction:

1.1 Advancement of oral vaccines Vaccination has saved countless lives since the 18th century.1,

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In addition to infectious

diseases, for which vaccines were initially researched, vaccinations are now used to prevent noninfectious, chronic, inflammatory and autoimmune diseases, such as atherosclerosis, Alzheimer’s disease, and cancer.3-5 The antigens used for vaccination have developed from attenuated live pathogens, such as viruses, bacteria and their recombinant derivatives, to the current classes with less toxicity and virulence reversion risks, such as antigenic subunits of viruses and bacteria, DNA, RNA and peptide vaccines.6 Currently, conservative injective vaccines are commonly administered in most countries but are complicated by the risk of needlestick injury, difficulties in self-administration and insufficient immunity inducement on mucous membranes.2, 7 Oral vaccines have shown robust immune response in both the small and large intestines and are highlighted for practical application in recent years. For example, the mucosa of the large intestine, which has fewer digestive enzymes than the small intestine, is an ideal site to induce both rectal and vaginal immunity, and intracolorectal (i.c.r.) vaccination at the large intestinal mucosa may induce effective cellular and humoral immune responses in the regional lymph nodes, in contrast to vaccination at a distant mucosa (e.g., intranasal) or by a parenteral or subcutaneous route. The intrinsic advantages of intestinal immunity and the improvement of both the efficiency and safety of the antigens are prerequisites for developing oral vaccines with the potential to stimulate a strong immune response with low invasive risk in a convenient process.8, 9 Except for sublingual immunotherapy aimed at desensitization, the versatile functions of synthetic antigen conveyers today makes it possible for oral vaccines to escape the GI tract,


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which is characterized by a powerful digestion system of proteases, unsuitable pH, and a myriad of live microorganisms that have deleterious effects on vaccines.2, 10 Most licensed oral vaccines and those undergoing renewed manufacture for licensure are attenuated viruses or bacteria and are capable of resisting the intestinal environment, replicating themselves and stimulating durative immune responses, for example, Rotateq®, Rotashield® and Tetramune® against rotavirus, Vivotif® against typhus, Dukoral® against cholera induced by Vibrio cholerae and enterotoxigenic Escherichia coli, and Kolera, CVD 103-HgR, known as Orochol® or Mutachol®, against cholera.2, 11-17 Attenuated viruses are efficient in most cases; however, there are inherent risks of reverting back to virulent pathogens, for example, the mutation of the virus in an oral polio vaccine caused an outbreak of poliomyelitis in Haiti and the Dominican Republic in 2000.18, 19 Thus, greater attention must be given to the security of orally administered vaccines. Nontoxic plant expression systems have demonstrated effectiveness as natural materials for the preparation of oral vaccines, such as the vaccine developed from hepatitis B major surface antigen.20 However, transgene expression in plants has disadvantages, including long-term growth and limited antigen expression ability. 1.2 Synthetic materials can be advantageous adjuvants for modern oral vaccines Adjuvants are molecules, compounds or macromolecular complexes that promote the potency and longevity of the specific immune response to an antigen, while causing minimal toxicity or long-lasting immune effects on their own.21,

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Modern vaccines receive protection and

synergism from adjuvants, such as bacterial factors (toxins, etc.), plant components (lectins, etc.), synthetic materials (polysaccharides, etc.), cytokines, epitopes and immune-related factors, including Toll-like receptor (TLR) agonists.21-23 According to their composition, sources, physiochemical properties, functions and mechanisms, these adjuvants can be classified as


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vehicles and immunostimulants. Although both can enhance the immune response, vehicles act by delivering vaccines to the target site and controlling their release, while immunostimulants regulate the immune system directly. One merit of vehicles is that they usually encapsulate vaccines and thus have the ability to protect the antigens and boost durative immune responses through a controlled response or targeted release of antigens.22 With the ability to integrate the functions of vehicles and immunostimulants, synthetic adjuvants are unique from the aspect of rational design for diverse structures, resulting in versatile functions, such as intelligent release and targeting ability, as well as the capacity for loading and delivering multiple effectors. Considering the harsh environment in the GI tract, which is a major liability of modern oral vaccines, the criteria for candidate adjuvants for oral administration are strict, including not only normal chemico-physical and physiological properties, such as size, branch degree, capacity for encapsulation, controlled release and toxicity, but also the ability to protect vaccines (i.e., antigens, effectors and the adjuvants themselves) in unwanted environments and to boost the specified level of immune response.24 Synthetic biocompatible nanoemulsions/particles and microspheres/particles have been employed as vaccine adjuvants to induce systemic and local immune responses during oral administration.22,

25, 26

Therefore, synthetic materials can

contribute to oral vaccination through optimized design to improve transport ability, stability/antidegradation performance, targeting properties, and immune-boosting efficacy. Scientists have developed strategies for oral vaccination that consider the physicochemical characteristics of both the adjuvants and the antigens. As a common strategy for oral vaccination, biocompatible carrier adjuvants should balance structural stability and controlled release, as well as the ability to protect the antigens from functional loss due to degradation in the GI tract. Frequently, the adjuvants are modified with their specific components at the outer surfaces or in


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the inner spaces to target distinct sites (Figure 1). Since protection of the target ligands, antigens and effectors and the controllable release of antigens must all be considered, intelligent response of the carrier adjuvants to the complex GI environment has become a research focus. These adjuvants can usually stimulate efficient phagocytosis of the antigens, and some polymer-antigen nanoparticles (20-100 nm) have been demonstrated to be phagocytosed efficiently by antigenpresenting cells (APCs) and to be retained in lymph nodes for a period of time without causing significant toxicity.27 The boosted immune responses caused by the transported antigens indicated the success of adjuvant-assisted vaccination. Although there are diverse strategies for adjuvant-payload combinations, synthetic materials integrating both a delivery system and immune enhancer can act as carrier adjuvants. As representatives, several classic or potential synthetic carrier adjuvants will be introduced, and their mechanisms and prospects will be described. 2. Organic polymeric adjuvants As early as 1988, avridine, a synthetic lipoidal amine incorporated in liposomes, was studied for its adjuvant activity in mice immunized orally with killed influenza virus vaccine (A/PR/8/34, H1N1) and was found to enhance remote-site IgA antibody response in the respiratory tract without side effects.28 Some organic monomers are potential immunostimulatory adjuvants; however, organic polymers are more attractive for oral vaccination because of their efficiency to deliver intact antigens and their ability to directly enhance immune response.29 Attributed to the biocompatible elements (oxygen, hydrogen, carbon, etc.) and chemical clusters (ester bonds, glucosyl groups, etc.) that share substantial similarity with natural organic molecules, synthetic organic materials are a pivotal choice for vaccine manufacturing due to their unsubstitutable biocompatibility. A variety of synthetic polymers are frequently used for administration as drug


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vehicles or vaccination adjuvants, including poly(lactic acids) (PLA),30 poly(D,L-lactide-coglycolide) (PLG),31 poly (lactic-coglycolic acid) (PLGA),32 poly(methylmethacrylates),33 and poly(alkyl cyanoacrylates).34, 35 Some are available orally, among which polymerized PLG and PLGA were involved in the earliest studies on the efficacy of nanoparticles (NPs) as adjuvants in oral vaccination after the successful use of natural liposomes and lipopolysaccharides as adjuvants for oral vaccination.36-40 Some of these adjuvants were demonstrated to be safe and efficient, with outstanding performance and application. 2.1 Adjuvants comprising classical linear polymers 2.1.1 PLA microspheres Based on lactic acids with meritorious biocompatibility and active chemical groups, poly(D, Llactic acid) polymers are among the most widely used synthetic biomaterials as vaccine adjuvants for drug transportation within the GI tract.41, 42 As demonstrated both in vitro and in vivo, PLA is degradable and is phagocytized by immune-associated cells. A PLA-entrapped rotavirus oral vaccination recently showed immunity enhancement, with long-lasting IgG and IgA antibody titer.41 The current issue concerning PLA particles is instability in the GI tract, which has led to attempts for external decoration that enables durative accommodation of peptide/protein antigens. 2.1.2 PLGA-based particles PLGA is one of the most popular biocompatible and biodegradable synthetic copolymers for clinical use in humans, and it is approved by the U.S. Food and Drug Administration (FDA) as a pharmaceutical excipient.43 PLGA or PLGA-derived nanoparticles or microparticles can be prepared by emulsification-diffusion, solvent evaporation and nanoprecipitation, and antigens


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can be encapsulated in the particles or attached to their surfaces by covalent conjugation or noncovalent interaction, such as electrostatic interaction, ionic bonding and hydrophobic interaction.32, 44, 45 A variety of antigens, such as bacterial lysates and toxoids, proteins/peptides, and DNA plasmids, can be effectively delivered in PLGA or PLGA-based particles with their structural and antigenic integrity fully maintained. In addition to its ability to enhance immunity, PLGA has adequate biocompatibility and excellent cell membrane penetration activity, as well as the ability to enhance internalization by APCs, such as macrophages and dendritic cells (DC). Modification of PLGA particles may improve the targeting ability or immune efficacy of systems, for example, CKS9 peptide improved the ability of PLGA vehicles to target M (microfold) cells.46-48 Since the initial application of PLGA as adjuvant for OVA and HIV at the end of last century,49, 50 studies have continued to implement PLGA as an adjuvant for oral vaccination. For example, PLGA nanoparticles loaded with Helicobacter pylori lysates, rather than free lysates, induced significant H. pylori-specific mucosal and systemic immune responses in BALB/c mice.51-53 The influence of the PLGA particle size on the immune response in female BALB/c mice was revealed by delivering 200, 500 nm and 1 μm PLGA particles orally with bovine serum albumin (BSA) entrapped as a model antigen. A higher serum IgG antibody level and a similar IgG2a/IgG1 ratio were observed with the 1 μm particles compared to the nanoparticles.54,

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Through surface modification, vaccines can be designed with different vector degradation and antigen release rates, resulting in varied levels of immunity response.55 PLGA particles have become one of the most widely used adjuvants for vaccines administered by different routes, and their potential as universal carrier adjuvants in modern oral vaccination is being studied. 2.1.3 PEG-PLA copolymer-based adjuvants


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The success of PLA nanoparticles and microparticles in oral immunization has demonstrated their potential as vaccine adjuvants. In some cases, however, plain PLA nanoparticles are aggregated within the GI tract and, partially due to the enzymes, rapidly lose the encapsulated antigen and fail to generate an effective immune response through oral vaccination. Originating from PLA, PEG-PLA copolymers (AB, ABA and BAB) were recently synthesized by ringopening polymerization for application as oral adjuvants.56 In the form of nanoparticles, hepatitis B surface antigen (HBsAg) was encapsulated by these copolymers through double emulsion solvent evaporation for administration in a mouse model. Compared with PLA nanoparticles, the slightly larger PEG-PLA copolymeric nanoparticles showed enhanced stability that allowed maintenance of particle segregation in simulated gastric and intestinal fluid. In addition to shielding the surface from unexpected reactions with the enzyme, the PEG ligands improved the hydrophilicity of the PLA nanoparticles and stimulated efficient uptake of copolymeric nanoparticles by gut mucosa. Compared with AB and ABA copolymers, BAB (PEG-PLA-PEG) copolymers showed the highest release and uptake by the gut mucosa. This ability is associated with the elevated hydrophilicity of nanoparticles provided by the PEG ligands. Due to the activity of PEG-PLA–based adjuvants, oral administration of copolymer-encapsulating-antigen (HBsAg) nanoparticles boosted the effective level of immune response, as demonstrated by the humoral immunity and mucosal (sIgA) and cellular immune response. The SIgA titer in all tested mucosa was significantly enhanced by copolymeric nanoparticles other than PLA nanoparticles, and BAB nanoparticles generated the most significant IgG titer. Comparison between polymeric nanoparticles and alum-HBsAg revealed stimulation of sIgA and IgG2a (the hallmark of TH1, indicating cellular immune response) by the former system and effective IgG1 by both. BAB nanoparticles containing the highest PEG proportion exhibited the most significant IgG2a titer.


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The IL-2 and IFN responses were significant with copolymeric nanoparticles compared to PLA nanoparticles. Therefore, PEG-PLA–based copolymeric nanoparticles represent a branch of modernized immunoadjuvants for oral immunization. 2.2. Cross-linking materials 2.2.1 Starch-based porous microspheres Microparticles/microspheres derived from starch are a family of biomaterials used for efficient drug and antigen transportation. Biodegradable microparticles comprising crosslinked polyacryl starch (PAS microspheres) originating from hydroxyethyl starch were initially synthesized as carriers for proteins (e.g., enzymes) and low molecular weight substances (e.g., drugs), and their applications in oral vaccination were studied.57 As starch-derived carriers for the encapsulation and controlled release of antigens, crosslinked polyacryl starch microparticles with pore structures were developed for oral vaccination. They can be prepared by polymerization of acryloyl glycidyl ester-modified starch (acryloylated starch) in a water-in-oil emulsion.58, 59 Polyacryl starch microparticles and other polymer-grafted starch microparticles have been successively developed as carrier adjuvants of oral vaccines. Through covalent linkage to polyacryl starch microparticles, the model antigen human serum albumin (HSA) stimulated strong DTH contributed-cellular response and IgM/IgG participated-humoral response in mice without causing detectable tolerance, and strong mucosal response, indicated by antigen-specific IgA, was detected compared with soluble HSA, which showed no efficacy.59 Additional starch microspheres were synthesized through grafting with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS). The microspheres entrapped HSA and boosted efficient immunity through oral administration by stimulating elements of the mucosal immune system,


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which includes antibody secretion in Peyer's patches (PP), antigen-specific lymphocyte reactivity from PP to splenic tissue, and enhanced numbers of spontaneous Th2-cytokine secreting lymphocytes disseminated from mucosal to systemic lymphoid compartments.60 Compared with administration with soluble HSA, the enhanced immune response toward TS-PDMS microparticles could be relevant to the controlled release of antigens and protection from entrapment to decelerate degradation in the GI tract. These starch-based oral vaccination systems are competent participants in mucosal and systemic vaccine delivery systems. 2.2.2 Cross-linkable polyphosphazene Due to the ability to form pH-sensitive hydrogels and membranes through cross-linking with multiple ions, such as calcium and aluminum, poly[di(carboxylatophenoxy) phosphazene] (PCPP) is a remarkable immunoadjuvant with a well-defined structure, biocompatibility, pH-dependent stability and synthetic origin. It is a prominent candidate for oral vaccination, with stability in neutral to acidic aqueous conditions, becoming unstable as the cross-linking process reverses in basic conditions.61, 62 With some sodium salt PCPP types currently undergoing clinical trials in humans, polyphosphazene derivatives with ionic groups have demonstrated excellent immunomodulating potential and are capable of enhancing immune responses against multiple bacteria and virus antigens, such as recombinant HBsAg (Figure 2). A novel polyphosphazene polyacid, poly(di(sodiumcarboxylatoethylphenoxy) phosphazene) (PCEP), was synthesized by macromolecular substitution, and with the appearance of amines, microspheres were achieved in aqueous solution by ionic complexation.63 Co-delivering PCEP with influenza X:31 antigen (prepared from purified influenza X:31 virus (A/Aichi/68 H3:N2)) through the oral route in BALB/c mice induced IgA, IgG1 and IgG2a titers, although the titers were detected at a relatively lower levels than those induced by intranasal (IN) or subcutaneous


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(SC) immunization. As a novel cross-linking adjuvant, the PCEP system is promising for oral immunization.64 2.2.2 Polymers that form hydrogels Highly absorbent hydrogels can be composed of either natural or synthetic polymeric networks. In contrast to the common cross-linkable molecules, some polymers can form hydrogels with high water contents (solvent) and the ability to prevent further water intrusion. Compared with natural hydrogels, such as agarose, alginate, methylcellulose and hyaluronan for drug delivery and tissue engineering,65 synthetic hydrogels are more flexible and more easily manipulated to obtain an accurate design, excellent compatibility, suitable degradability, and environmentsensitive (pH, temperature, etc.) release. The first reported synthetic hydrogel for loading antigens was poly(methacrylic acid), which was administered orally to sheep.66 Due to its optimal size (5 mm in diameter, 3 mm in length) and density (specific gravity of 1.4), the hydrogel bypassed the first stomach (rumen) and released antigens in the lower GI tract by swelling. Exploration of synthetic hydrogels for oral vaccination is in progress. Recently, mannan-loaded biodegradable thermosensitive poly(ε-caprolactone)-poly(ethylene glycol)poly(ε-caprolactone) (PCL-PEG-PCL, PCEC) was synthesized as an injective adjuvant and was shown to mix with basic fibroblast growth factor (bFGF) at low temperature to form a nonflowing gel at body temperature in situ and to further control the release and enhance the immunogenicity of bFGF.67 Synthetic polymeric hydrogel adjuvants have strong potential in the application of oral vaccines. 2.3 Branched polymers


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Polymers with branched structures can integrate the desirable properties of multi-chemical chains and groups into a single molecule to improve several characteristics, such as dispersity, drug loading capacity, stability, and protection level. A successful construct, sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide) (SB(43)-PVAL-g-PLGA), was endowed with antigen affiliation capability.68 In contrast to common encapsulation, the antigen, tetanus toxoid (TT), was adsorbed onto the surface of NPs with surface charges to preserve their biological activity and improve antigen performance. An immunization experiment demonstrated that both oral and intraperitoneal administration of TT-loaded NPs efficiently increased IgG and IgA serum titers, revealing the potential use of branched SB(43)-PVAL-g-PLGA as a mucosal vaccine adjuvant. Tree-like dendrimers with repetitive branches have spherical three-dimensional morphology with structural perfection. Dendrimers are usually symmetric and highly monodisperse. A typical dendrimer has three major portions: a core, an inner shell and an outer shell. Each portion can be functionalized with properties such as solubility, thermal stability, biocompatibility, and better sizing and loading capacity for specific compounds. Dendrimers can be synthesized by divergent synthesis extending outward from a multifunctional core, convergent synthesis proceeding inward from surface of the sphere to the core, and other methods. Dendrimers designed with various surface and internal functionalities have been applied to drug and gene delivery, sensors, blood substitution and diagnosis and therapy. By tailoring the functional groups within the cores or on the branches and surfaces, dendrimers have been developed for vaccination applications,69-74 for example, tetragalloyl-Dlysine dendrimer (TGDK) and poly(amidoamine) (PAMAM) dendrimers.75, 76 With the ability to target M cells, TGDK was produced by reacting Fmoc-D-lysine dendrimer resin and 3,4,5-


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trimethoxybenzoic acid chloride, followed by treatment with boron tribromide.75 Oral administration of TGDK conjugated with rhesus CCR5-derived cyclopeptide (the antigen) in rhesus macaque resulted in a significant increase in stool IgA response and induced neutralizing activity against SIV infection. Derived from divergent synthesis, PAMAM is composed of highly branched dendrimers polymerized through generation from an initiator core, such as ethylene diamine or ammonia (Figure 3).76 With increased generation, the shape of PAMAM shifts from a planar ellipse to a spherical conformation with a hydrophobic interior space capable of transporting payloads.77, 78 PAMAM dendrimers have been patented as adjuvants for influenza pneumonitis vaccination. Their mid generations (e.g., Generation 6) boosted antigenic response and enabled the use of low levels of antigens and dendrimers.76, 79 PAMAM dendrimers may be beneficial to opening the tight junctions of epithelial barriers, and this merit has been applied to the transportation of oral drugs.80 There are still challenges to maintaining its stability in the GI tract and the blood stream, and strategies based on decoration may be beneficial for improving the relevant properties. Advances in the understanding of dendrimers, their architecture, physicochemical properties, and behavior in vivo have led to their application in modern vaccination, and more achievements are expected in the future. 2.4 Self-assembled adjuvants 2.4.1 Micelles Self-assembled micelles are aggregated forms of amphiphilic molecules in a liquid colloid that usually have polar head regions in contact with the surrounding solvent, sequestering the hydrophobic single-tail region in the micelle center. Micelles can assemble with drugs in


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aqueous solutions. Nonpolar solvents are suitable for the assembly of micellar systems carrying hydrophilic compounds or water-soluble proteins, such as antigens.81 By encapsulating baculovirus displaying influenza hemagglutinin within the reverse micelle structure of phosphatidylcholine and delivering the resulting En-BacHA into the gastrointestinal tract of mice, enhanced HA-specific serum IgG and mucosal IgA antibodies were boosted, and 100% protection against 5 MLD50 of the HPAI H5N1 influenza strain was conferred on vaccinated mice. In contrast to conventional emulsions, baculovirus displaying HA is solubilized in the oil phase and encapsulated within the polar head groups of phosphatidylcholine, while the hydrophobic tail faces the continuous oil phase. This anhydrous encapsulation prevents the antigen from denaturation during gastrointestinal transit upon oral vaccination.82 2.4.2 Lipid vesicles 2.4.2.1 Liposomes A liposome can dissolve hydrophilic solutes in its aqueous solution core, and the hydrophobic bilayer membrane associates with hydrophobic chemicals. Contents loaded in either form are not readily released. Through designs with a different isoelectric point (pI), liposomes can release their contents at a specific pH when the liposomes and drugs are neutralized. Liposomes are widely used for drug encapsulation and for mediation of transgene expression. Due to the enhanced protection of many kinds of oral drugs and the ability to stimulate cell internalization, liposomes have become candidates for oral vaccines.83 Through encapsulation of vaccines/antigens, a mucosal response to microbial polysaccharide antigens and other antigens was successfully achieved.84-86 Despite the ingredient loss caused by chewing, swallowing and saliva flowing, antigen-loaded multifunctional liposome-constituted microneedle arrays


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(LiposoMAs) administered to mice through the oral mucosa induced mucosal immunoresponses against the loaded antigens, accompanied by a mixed Th1/Th2 immunoresponse and strong cellular/humoral immunity due to special adjuvanticity and targeting delivery functions of the nanoparticulate VADS.87 Intact liposomes were endocytosed by M cells, which delivered the antigens to the underlying lymphoid cells in the Peyer's patch.88 Due to the convenience of developing liposome-encapsulating-antigen vesicles via assembly and the flexibility of tailorable liposome derivatives, liposomes functionalized with DSPC (distearoylphosphatidylcholine) and cholesterol,89

1,2-dioleoyl-3-trimethylammonium

propane

(DOTAP)-based

liposomes,90

liposomes coated with an inner layer of tremella and an outer layer of acid-induced alginate,91 and covalently membrane-bound UEA1 targeting polymerized liposomes (Orasomes, approximately 200 nm diameter) to intestinal M cells have been established for enhanced or M cell targeted oral immunization in recent years.92 2.4.2.2 Bilosomes Vesicular carriers such as liposomes and niosomes have potential for oral vaccination but are unstable in the gastrointestinal environment. Bile salt is produced in the liver and stored in the gallbladder and is formed through conjugation of bile acids with taurine or glycine. Stabilized by bile salt, bilosomes represent a key advance in oral vaccine delivery because they are more resistant to disruption by gastric acid and enzymes.10, 93 The incorporation of bile salts into lipid vesicles (bilosomes) successfully induced mucosal and systemic immunity via the oral route. Different formulations of physically modified bilosome oral vaccines could enhance the effectiveness of candidate vaccine antigens.94 Modification of bilosomes provides several approaches for oral vaccination. For example, glucomannosylated bilosomes may be suitable for oral mucosal vaccination,95 and cholera toxin


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B subunit conjugated bilosomes can deliver recombinant hepatitis B surface antigen to M cell targets.96 2.5 Adjuvants based on charged polymers Charged polymers introduced as coatings or modifiers can confer unique surface properties to vaccine vesicles.97 In addition to achieving well-dispersed and stable suspensions of nano- to microparticles, some particles decorated with cationic polymers, such as polyethyleneimine (PEI) and chitosan, were endowed with improved mucoadhesive properties conducive to enhanced immune responses.98 Coating vaccine systems with poly dimethyl diallyl ammonium (PDDA), PEI and chitosan can delay the appearance of the peak IgG titer and elevate the IgA titer, suggesting the potential of these coatings for mid-to-late immune responses through mucosal vaccination.97 With the ability to entrap proteins, calcium-alginate and calcium-yam-alginate microparticles can be coated with N-(4-N,N-dimethylaminocinnamyl) chitosan to elevate both IgG and IgA titers. These coated microparticles showed strong mucoadhesive ability and sustained antigen release.98 Due to interest in vectors for oral vaccines, we recently developed a pH-sensitive PMMMA nanolayer to encapsulate the inner PLGA/antigens NPs using poly(methacrylic acid) (MAA) harboring carboxyl groups, methyl methacrylate (MMA) and methyl acrylate (MA) as precursors for free radical polymerization at the surface of PLGA NPs.99 The PMMMA-PLGA/TRX-SIP oral vaccines stimulated a high level of specific immunoglobulin (IgM) and durative immunoprotection in a tilapia model against group B Streptococcus (GBS) disease. These nanoparticles have uniform morphology in aqueous solution, where PLGA nanoparticles are prone to aggregate due to the hydrophobicity of their polyester constituents. Weak alkaline conditions leading to ionization of the -COOH in the MAA blocks in PMMMA could cover the


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nanoparticles with repulsive negative surface charges. Increasingly alkaline conditions activated the pH-responsive phase transition of PMMMA. In contrast to the size effect, which facilitates the escape of microparticles from cell uptake in the small intestine, this negative charge repulsion shields the PLGA/antigens nanoparticles from adhering to the mucin/epithelial cells in the small intestine and makes it possible for the PMMMA-protected nanoparticles to reach the large intestine, which has higher pH values, followed by active absorption of the PLGA/antigen nanoparticles by the target immune cells, thereby promoting efficient presentation of the antigens.100 3. Porous inorganic particles Most current “soft” materials undergo diffusion or degradation in the GI tract. Porous inorganic materials have great potential for modern oral vaccination due to the minimal effect of enzymes and pH fluctuations on their stability and antigen loading patterns, including covalent and noncovalent conjugation. Porous silicon, carbon NPs and microparticles are more effective for oral vaccination than other inorganic materials. The synthesis of inorganic porous materials using structure-directing polymers has made significant progress in the control of the morphology and function of the adjuvants. With regard to the instability demonstrated by traditional calcium- and aluminum-based adjuvants for the parenteral route, synthesized porous silica and carbon materials have several merits, such as enhanced safety, consolidation, designable antigen loading capacity and release profile that depends on tailoring of the architecture, such as proper particle scale, pore size, surface charge, hydrophobicity, and dispersivity.101-104 Although problems associated with biocompatibility occur with most inorganic materials, several silica- and carbon-derived porous systems have demonstrated security for oral administration.97


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3.1 Silica nano- and microparticles Silica nano- and microparticles provide immunogens with defense against the harsh GI environment by shielding them within their pores. One silica-based nanoparticle used for oral vaccination is porous SBA-15 silica.105 Polymeric SBA-15 nanocylinders can be prepared by using organic structure-directing agents, for example, the amphiphilic block copolymer poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO-PPO-PEO). These particles are approximately 2 μm in length and have internal, hexagonally ordered mesopores (mean diameter of 10 nm) (Figure 4).106 Calcination at 500°C generated porous structures with large interlattice d spacings of 74.5 to 320 Ǻ between the (100) planes, pore sizes from 46 to 300 Ǻ, pore volume fractions up to 0.85, and silica wall thicknesses of 31 to 64 Ǻ. The silica wall thickness and pore size can be adjusted by varying the heating temperature (35C-140C) and time (11 to 72 hours). Using a variety of poly(alkylene oxide) triblock copolymers and adding cosolvent organic molecules, SBA-15 can be prepared with wide range of uniform pore sizes and pore wall thicknesses at low temperature (35°C to 80°C). A thermally stable product can be achieved by removing the organic polymers with ethanol or heating at higher temperature. Through in-depth investigation, SBA-15 achieved successful application in immunization by both the intramuscular and oral routes due to the specific physical and structural properties. SBA-15 has the ability to evoke immunological memory, and the antigens can be entrapped into the silica pores to produce a sustainable vaccine system.105, 107 In addition to SBA-15, two porous silica nanoparticles, S1 and S2, were reported as adjuvants for oral vaccination.107 The highest levels of IgG and IgA titers were induced by S1 NPs (430 nm) loaded with BSA, followed by S2 (130 nm) and SBA-15 NPs loaded with BSA (1-2 m in length and 0.4 m in diameter). BSA-loaded nanoparticles induce both T-helper 1 (Th1) and T-


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helper 2 (Th2) mediated responses through oral immunization. These nanoparticles are chemically stabile and biocompatible and possess adjuvant effects.107 Modification with organic polymers or biomaterials, such as chitosan, improves the properties of the organic-inorganic hybrid systems. Under simulated gastric conditions, the chitosanhydrophobic silica composite Aerosil R972 microspheres showed lower water affinity, better thermal degradation and stability in acidic conditions, as well as the ability to retard the release of rifampicin (drug model), inspiring further investigation as a carrier adjuvant for oral administration.108 3.2 Carbon particles Carbon nanoparticles, such as nanotubes (NTs), are attractive for current medicinal applications. Carbon-derived NPs are challenged by cytotoxicity and insolubility in aqueous media.109, 110 The rational design and modification of carbon nanoparticles can endow them with advanced characteristics for immune applications. Based on the hydrophobicity of carbon NPs, carbon nanoparticles (C1; 470 nm) were synthesized to prevent the degradation of antigens in the GI tract and to optimize transportation to M cells. The mesoporous C1 nanoparticles were prepared using a silica template and sucrose as the carbon source. The pore sizes were 40-60 nm, providing good capacity for antigen encapsulation. C1 loaded with BSA stimulated a high IgG titer. Mucosal IgA was detected in intestinal, salivary and vaginal secretions, indicating effective stimulation of the mucosal immune response. The Th1 and Th2 mediated responses were elicited simultaneously.111 As a natural mineral, inorganic CaCO3 microparticles have unique advantages, such as biocompatibility and porous structural characteristics, making them suitable for the protection and controlled delivery of antigens within the GI tract. On this base, self-adjuvanting peptide


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nanofiber-CaCO3 composite microparticles were prepared for oral vaccine applications. By dissolving OVA-KFE8 nanofibers in CaCl2 and Na2CO3 solutions, OVA-KFE8/CaCO3 composite microparticles were achieved. OVA-KFE8 nanofibers were encapsulated within the CaCO3 cores, with only a small portion (15%) released after 5 wash cycles. Oral vaccination using the composite microparticles stimulated efficient penetration of the mucus barrier to immune inductive sites, followed by mucosal antibody responses, particularly the protective IgA isotype.112 Due to the capacity for protecting and transferring antigens along the GI tract, tailorable inorganic materials are a promising branch of adjuvant conveyers that may significantly influence modernized vaccination. 4. Paths and mechanisms of adjuvant-enhanced oral vaccination Oral vaccines with a non-invasive nature and higher compliance for all ages are suitable for eliciting local mucosal immunity to combat mucosal pathogens and have potential to become safe and convenient substitutes for conservative vaccines. However, the harsh gastrointestinal environment, the selective permeability of the mucus barrier, and the trans-membrane delivery of antigens are challenges to the success of oral vaccinations. Synthetic adjuvants are an attractive way to solve these problems because of their feasibility to be designed and modified through polymerization to achieve higher levels of immune response. The gastrointestinal tract is vital for balanced nutrient absorption and immune protection. The intestinal immune system plays an important role in guaranteeing effective oral vaccination with the assistance of well-designed adjuvants. Both the small and large intestines are efficacious targets for oral vaccination since abundant immune cells are distributed among the intestinal epithelial cells.


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In the human body, the intestine possesses the largest amount of lymphoid tissues,113 known as gut-associated lymphoid tissues (GALT), comprising the PP in the small intestine, the appendix, the lymphoid follicles in the large intestine and rectum, lymphoid cells and plasma cells throughout the lamina propria of the gut, and lymphoid tissues in the esophagus, stomach, and Waldeyer's ring, composed of adenoids and tonsils. PP and other immune-associated cells (e.g., lymphocytes and macrophages) are common targets for oral vaccination. PP are located within the ileum of the small intestines, belong to the lymphatic system and are an important component of the digestive tract's local immune system. They present antigens in the digestive tract to the immune system, which initiates a non-specific innate immunization followed by a long-lasting specific adaptive immunization in vertebrates. Then, the immune system evolves to recognize and neutralize pathogens and creates an immunological memory for quick immunization against this pathogen each time. Microfold cells (or M cells) are found in the GALT of the Peyer's patches and in the mucosaassociated lymphoid tissue (MALT) of other parts of the gastrointestinal tract. These cells initiate mucosal immunity responses on the apical membrane of the M cells and allow transport of microbes and particles across the epithelial cell layer from the gut lumen to the lamina propria, where interactions with immune cells can occur (Figure 5).88, 114 For oral immunization using synthetic materials as adjuvants, this process is significant for the establishment of durative antigen-specific adaptive immunization, also known as the acquired immune system, including mucosal, humoral and cell-mediated immunity components, in which a series of highly specialized, systemic cells and processes are involved. Some examples include presentation of the foreign antigens by APCs, including dendritic cells, B cells, and macrophages with special "co-stimulatory" ligands to T cells expressing co-stimulatory receptors;93 antigen presentation


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through major histocompatibility complex (MHC) molecules (known as human leukocyte antigen (HLA)) to T cells such as CD4+ T-helper cells;115 clearance of different pathogens by cytotoxic T cells and Th1 or Th2 type response;87, 107 the antibody responses and cell-mediated immune responses performed by B cells and T cells lymphocytes, respectively;97 the somatic hypermutation and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments), which enable adaptability of the system, resulting in the unique expression of antigen receptors on each individual lymphocyte;116,

117

and development of

immunological memory managed by memory B cells and memory T cells (Table 1).105, 107 Targeting vaccines to the small intestines is a common strategy for oral vaccination. The adjuvants must protect the vaccines from the harsh intestinal environment, which is filled with proteases, esterase, trypsin and other enzymes, as well as commensal flora. Although success has been achieved in the targeted delivery of oral drug systems,118, 119 oral vaccines with targeting ability are less common.8 Both vectors and antigens are challenged by digestion or inactivation before cell uptake within the small intestine. Since an abundance of enzymes reduces the amount and activity of antigen, adjuvants, such as nanoparticles based on biomolecules, are expected to be protective and capable of stimulating antigen presentation through activation of cell absorption of the antigens, which is mediated by transporters.120 In this situation, they are preferred adjuvants for cell internalization, with optimized bio-utility and biodegradability for the release of the antigens, which are either encapsulated by themselves or other inner adjuvants. Due to this property, they are frequently impaired before cell internalization of the antigens in the small intestine. This is the main challenge to increasing the efficiency of most oral vaccines with synthetic adjuvants, including most biodegradable polymers, such as those based on PLA,


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PLGA, lipid, polysaccharides, peptides and all the other molecules comprising biodegradable chemical groups. The large intestine is suitable for oral vaccines due to its close relationship with immunity and lower concentration of enzymes.121 The immune responses (both cellular and humoral) induced by intracolorectal (i.c.r.) vaccination occur in regional lymph nodes of the large intestinal mucosa, an optimal site for both rectal and vaginal immunity.122 Because vaccines enter the large intestine, where the pH varies significantly, after they pass through the small intestine, the increased complexity of the environment increases the difficulty for tailoring the adjuvants. During the course of providing protection to the antigens in both the highly acidic gastro and enzyme-abundant small intestine, pH sensitivity and small intestine escaping activity are the basic requirements for these adjuvants to target the large intestines and release antigens there. Although less common, several adjuvants with pH sensitivity have been studied for application in large intestine-targeted oral immunization based on their pH-induced stability under gastro and small intestinal conditions.8 Zhu. et al. reported that the nanoparticle uptake primarily in CD11b+B220int macrophages and secondarily in CD11c+CD11b+ dendritic cells (DCs derived from bone marrow) in the lamina propria of the large intestine and antigen-specific T cell responses were induced. As indicated by the antigen-specific tetramer positive CD8+ T cells detected in the large intestine, colorectal immunity was successfully induced by an oral FS30Dcoated vaccine.8 In our recent study, a robust antibody (IgM) response was found in tilapia after oral immunization with PMMMA-PLGA/TRX-SIP.99 The properties of the intestinal tract and adjuvants determine the behavior of oral vaccines. Particle size influences the stability and speed of antigen release and determines the efficiency of cell uptake.56 For example, the small intestine is unable to absorb microparticles (1 m), so a


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large size may help the vaccine reach the large intestine.8 In our study, a PMMMA outer nanolayer, which is biocompatible but not preferred by the epithelial cells lining the small intestine, could enter the large intestine.99 By using tailorable adjuvants, the vaccine size and ability for cell internalization can be controlled to target different intestinal positions. In addition, the particle density, charge, utilization and biodegradation properties, immuneassociated effectors, target ligands of the adjuvants and specific target objects (or receptors) in the intestines affect the position and efficiency of antigen uptake, presentation and consequently, the immune response (Table 1).8, 56, 66, 92 Some reported adjuvants boost the systemic, mucosal and cellular immunostimulatory responses following oral administration, for example, chitosan-glucomannan nanoassemblies (sCh-GM-NAs) increase the concentration of mannose molecules over the surface of NPs, which may ultimately synchronize precise target delivery to the antigen-presenting machinery.123 Positively charged chitosan can help vaccines to penetrate the enzyme-abundant mucus layer and the tightly lined epithelial cells to enhance cell presentation. In addition to the protective activity of synthetic adjuvants, an increased level of antigen uptake by targeted immune cells can be achieved through the cooperation of utilizable biomaterials or chemical groups.95 Although less common, some adjuvants are directly associated with immunity. For example, SBA-15 can evoke immunological memory through oral administration. These characteristics are linked to the specific physical and structural properties of adjuvants.105 5. Role of rationally designed adjuvants in oral vaccination It is highly desirable that the adjuvants for oral vaccines be designed with versatile functions to achieve targeted delivery of the antigens for presentation. Promoting the intensity and duration of the robust immune response is a criterion for adjuvant optimization. Rational design of the


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conformational plasticity and functional couplings of the adjuvants can improve vaccination performance.32 5.1 Functional enhancers Directional optimization of adjuvants is an efficient approach to rapidly screen functional adjuvants and reduces the time and economic expenses. 5.1.1 Stabilizers A stabilizing effect of antigens is necessary during oral vaccination considering the harsh conditions in the GI tract. Since antigens are vulnerable to the stomach acidic and enzymeabundance of the small intestine, synthetic materials can be designed to protect them. The majority of adjuvants used for oral administration are resistant to the gastric juice and small intestinal fluids in the GI tract. The degree of stability directly influences antigen intactness, success of targeted delivery, release velocity, and the intensity and durability of the immune response. PEG, a widely used ligand for drug delivery, can be used as a shield to prevent digestion of PLA adjuvants in the GI tract, resulting in enhanced mucosal uptake and satisfactory humoral immunity and mucosal and cellular immune response.56 5.1.2 Modulators of antigen maintenance and release The presentation of antigens to the immune system by APC cells is the basic procedure to develop immune protection. As a solution for the release and delivery of antigens to APC cells, the degradation of the adjuvants at simulated physiological conditions can be monitored by adjusting the ratios of each component with unique properties. Hydrophilicity and biodegradability can be regulated to control the equilibrium between the encapsulation and


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release of the antigens, further optimizing antigen delivery to the target immune cells and presentation to the immune system. Hydrophilic PEGs with various lengths and molecular weights can be used to modulate the water dispersion and shielding degree of vaccine systems, through which the contact between the inner vaccine and the enzymes can be regulated, and antigen release, followed by presentation, can be optimized. The release of CyA from PLA microspheres was increased by the addition of biodegradable constituents, such as fatty acid esters, to the adjuvants,30 and the chemically stable resin improved the durability of the vaccine in the GI tract. Environment-sensitive and phase-changeable adjuvants control the targeted release of antigens. This strategy is based on elaborate tailoring of materials. These adjuvants are highly stabile and provide strong protection of antigens from stomach acid. When they arrive at the target location in the small or large intestine or when they are internalized by immune cells, they initiate phase transition and release the antigens. The complexity of the GI tract conditions remains a challenge for most synthetic adjuvants. Zhu et al. developed pH-sensitive microparticles to target the large intestine (Figure 6). The methacrylate-based polymer Eudragit FS30D encapsulating PLGA microparticles (>10 μm) were expected to escape the small intestine due to their size since the small intestine absorbs particles up to 1 μm. The antigens remain intact and are selectively delivered to the lower GI tract, where the enzyme concentrations are much lower than in the small intestine.8 5.1.3 Cellular uptake stimulators Access of the oral antigen to the immune cells residing on the intestinal walls is essential for eliciting an immune response. Although peptide fragments with incomplete epitopes may be effective, the intake of intact antigens by immune cells can boost the immune response with


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increased specificity. Therefore, strategies associated with antigen encapsulation are being explored to facilitate cellular internalization of intact antigens. Adjuvants can be designed with biocompatible (or bioutilizable) resources, which mechanically prefer to be actively transported into immune cells via mediation of the transporters on the cell membrane.120 Polyesters, solid lipids, phospholipids, polysaccharides, glycol-based polymers and some small biomolecules are prominent candidates as adjuvants to accelerate cell internalization.124-127 The optimization of vaccines with targeting ligands can be used to deliver vaccines to the target immune cells, but cell uptake depends on the components and size of the vaccines. Generally, the particle size for cell uptake is less than 1 m in diameter and should not exceed 300 nm.125 Some vectors, such as PEI and liposomes, can stabilize and deliver DNA for antigen expression in immune cells.125, 128-130 These positively charged molecules have strong affinity to mammalian cells with net negative surface charges due to their phospholipid content. However, these polymers cause various degrees of injury to mammalian cells and are not ideal for single use in oral vaccination. Modification with biocompatible molecules has been used to attenuate the toxicity of these molecules through either covalent conjugation or noncovalent coassembly.129 The concentration of antigens is essential for cell absorption and presentation. Improvement of the encapsulation of antigens has long been a widespread concern. In this respect, the majority of current adjuvants, such as synthetic PLGA nanoparticles and porous silica particles, have shown sufficient capacity to transport antigens and maintain their immunogenicity. 5.1.4 Immunity enhancers Adjuvants comprising immune-enhancing molecules can remarkably improve specific immune responses.97 For some adjuvants that coat antigens, optimization of the release


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properties plays an important role. However, recent findings revealed that some coating approaches, for example, encapsulating porous carbon particles with poly dimethyl diallyl ammonium (PDDA), PEI and chitosan (CTS), exhibited similar release rates and delayed immune response (indicated by the IgG peak) but significantly induced the mucosal immune response (indicated by IgA titers) compared with BSA or BSA/FCA adjuvants.97 Direct modification of antigens is an effective strategy to enhance the immune response. With the assistance of mucosal carrier adjuvants (e.g., chitosan), fusing peptides, such as the cholera toxin (CTB), with the antigen peptides can stimulate greater specific humoral, cellular and mucosal immune responses.126 Adjuvants with mucosa adhesion capability (e.g., chitosan) and those with the ability for controlled release of antigens in the GI tract or immune cells (PLGA NPs et al.) can prolong the generation of the immune response and enhance the duration of immunity.98, 99, 120, 126 Some adjuvants can enhance the immune system through direct interactions. SBA-15 evokes immunological

memory,

which

is

essential

for

successful

vaccination.105

CpG-

oligodeoxynucleotide (CpG-ODN) was investigated as a candidate adjuvant due to its ability to enhance immunity through a series of procedures stimulating the function of immune cells, including dendritic cells, APCs, mononuclear macrophages and B cells, resulting in efficiently improved mucosal and systemic immune response. CpG-ODN can facilitate transepithelial delivery and therefore has potential as an adjuvant in oral vaccination.131 Other immune-related factors, for example, TLR ligands with the capability to induce synergistic activation of T cells, have received increased attention in recent years.132, 133 5.1.5 Target tracers


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It is highly desirable that adjuvants have the ability to recognize or automatically trace the target immune cells in the GI tract because a greater concentration of vaccine near the target cells can increase the frequency of cell uptake and presentation of the antigens before they are degraded in the GI tract. Target ligands can direct oral vaccines to their destination. M cell-homing peptide (CKSTHPLSC) is such a ligand that was discovered by phage display. CKSTHPLSC demonstrated high affinity to M cells and facilitated the transportation of chitosan nanoparticles.134, 135 Based on M cell-homing peptide fused antigens and thiolated pH-sensitive eudragit (TE), which can adhere to mucosa and enhance permeation, an efficient adjuvant was developed to deliver antigens to the ileum, which has abundant epithelial M cells.134 Oral delivery of TE microparticulate antigens resulted in efficient transcytosis of antigens through M cells, followed by strong protective sIgA and systemic IgG antibody responses. T cell immune responses and strong CD8(+) T cell responses were stimulated by the enhanced production of IFN-gamma in the spleen, demonstrating a combinatorial method for the development of M celltargeted mucosal vaccines. Cholera toxin (CT) can specifically affiliate with the ganglioside GM1 receptor on most cells. Particles conjugated with recombinant nontoxic pentameric B subunit of CT (rCTB) with specificity for GM1 can target the villus epithelium due to the GM1 binding capacity.136 Recently, multiple-mutated CT (mmCT) was engineered as a nontoxic mucosal adjuvant to enhance the immune response to co-administered mucosal vaccines.137 Ulex europaeus 1 (UEA-1) lectin is a ligand that can direct oral vaccines to the M cells of the PP to enhance the immune response.138 Some toxoids (e.g., the TT and the botulinum toxoid


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(BT)) have the ability to direct oral vaccines to M cells to enhance the antigen-specific serum IgG response and mucosa IgA response.139 Although the period of contact between antigens and enzymes in the small intestine can be shortened by accelerated delivery of antigens to APCs with the assistance of adjuvants, the effectiveness of small intestine-targeted vaccines is limited by the abundance of proteases and other enzymes in the small intestine. The latest strategy is based on large intestine-targeted vaccination by providing protection to prevent digestion of the antigens in the small intestine. These adjuvants are stable in the small intestine and are not absorbed by the small intestinal cells; thus, the adjuvanted antigens can bypass digestion in the small intestine and be delivered to the large intestine. Efficient release of the antigens can be achieved through phase transition of the adjuvants in the large intestine at increased pH. In general, the current large intestine-target adjuvants are based on the small intestine-bypass activity and the pH-responsive antigen release of the adjuvants.8,

99

In this

respect, the development of ligands with strong specificity for large intestine immune cells is expected. 5.1.6 Environment sensors Due to the extremely differentiated pH and enzyme conditions in each part of the GI tract, responsive antigen release at the targeted location requires adjuvants with environmental sensitivity. The majority of the early generation of adjuvants for oral vaccines had a single component providing protection to the antigens in the stomach. Currently, pH-sensitive hydrogels, resins, and surface membranes are used to protect the antigens in the stomach and small intestine to ensure release of the antigens or the inner vaccines (such as the PLGA/antigen based vaccines) in the large intestine. We recently explored a double-layered oral vaccine


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adjuvant.99

Its

outer

pH-sensitive poly[(methyl

methacrylate)-co-(methyl

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acrylate)-co-

(methacrylic acid)] (PMMMA) layer is not preferential for cellular uptake but endows the nanoparticles with pH sensitivity, supporting the survival of the nanovaccine system in the stomach and small intestine. The significantly increased pH of the large intestine can initiate a phase transition of the PMMMA nanolayer due to ionization of the –COOH groups, leading to alteration of PMMMA from hydrophobic to hydrophilic and resulting in the subsequent release of the PLGA/antigen inner vaccine particles (Figure 7). Due to the nanoscale of the PLGA/antigen particles and the active transportation of the PLGA NPs to the immune cells, the antigens achieve efficient presentation. A strong immune response was achieved in a tilapia model. Labeled antigens delivered orally by PMMMA-PLGA are accumulated in the large intestine and immune-related organs, which was not observed with antigens encapsulated by PLGA only. The pH sensing adjuvant constituents can enhance oral vaccines in many circumstances. 5.2. Intelligent adjuvants with integrative functions Traditional synthetic adjuvants with a single component lacked the necessary flexibility and adaptability to survive the dramatically differing stomach and intestinal environments and could not control antigen release at the target position. Modern adjuvants for oral administration combine the advantages of two or more components that are beneficial for accurate delivery of intact antigens to APCs. In addition, specific types of immune response can be stimulated with enhanced intensity and durability. Components that participate in different roles, such as encapsulating the antigens, targeted delivery, or enhancing cell internalization, can be integrated into a single optimized adjuvant group to increase immune efficiency and develop long-term immune responses.


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6. Development of synthetic adjuvants for oral vaccination 6.1 Challenges and difficulties of synthetic adjuvants for oral vaccination Despite the attractive features of synthetic adjuvants to overcome physiological obstacles, including the enzyme-abundant mucus layer and the tightly lined epithelial cells, which can prevent mucosal vaccines from reaching APCs, few oral vaccines are currently approved for human use.115 Several challenges exist, such as the difficult transposition of results from animal models to humans, injury to the antigens caused by organic solvents during microencapsulation,115 the high costs of the medical grade precursors and manufacturing of integrated adjuvants with multiple functions, and the necessity of minimal toxicity and optimized activity to stimulate immune responses. A network is needed to evaluate adjuvants, including their metabolism, in vivo cycling, potential influence on the organisms, the interaction between the adjuvants and the immune system, the long-term biocompatibility of the adjuvant, and the mechanism for improving the immune response through oral vaccination. 6.2 Outlook of the synthetic adjuvants for oral vaccination Depending on the physicochemical characteristics of the adjuvants, peptide-based vaccines have been optimized for efficient oral vaccination. Although still considered immature for oral administration, DNA and RNA vaccines have become the focus of modern oral vaccines. Because RNA is extremely sensitive to enzymes, oral vaccines based on RNA have not been successful. In contrast, although gene delivery strategies based on cationic biomaterials, such as PLA-modified or glycerol-conjugated PEI branched molecules, have shown potential for the mammalian cell expression of target antigens. Their security for oral administration must still be approved.140, 141 The current DNA oral vaccines include those developed on the basis of intestinal


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probiotics, such as Lactococcus lactis.142 This strategy is deemed relatively safe compared to those designed for expression within immune cells, which showed mutation risks.143 However, durative expression of antigens may induce problems related to immune tolerance. Colonization of recombinant bacterial strains within the GI tract is another issue. The combination of synthetic polymers with targeting ability and controlled degradation properties with antigen-expressing bacterial cells might provide a method for the development of a time-dependent system with the ability to anchor probiotics at the target loci, sustain a high level of antigen expression for an appropriate period of time, and terminate antigen expression by clearance of the objective probiotics from the GI tract. Based on current proteomics and solutions to issues related to gene recombination, the expression of nontoxic peptide antigens in immune cells has great potential. Correspondingly, the development of biocompatible DNA (or RNA) transfection adjuvants with targeted delivery ability within the GI tract has potential to improve the efficiency of nucleic acid vaccines. Tumor vaccination has become an important focus of recent investigations.144, 145 Due to the high frequency of genome mutation in malignant tumor cells, particularly those with resistance to antitumor drugs and those characterized by metastatic activity, immune therapy has shown potential to challenge tumorigenesis and cancer progression. Tumor-associated antigens (TAAs) have multiple functions in tumor cells, facilitating propagation and escape from apoptosis and shielding them from the immune system. TAAs are frequently expressed at much higher levels in tumor cells than in normal cells. However, most intrinsic TAAs on tumor cells are ineffective to elicit immune responses with specificity to cancer cells, either because of their relatively low abundance on tumor cell surfaces or the lack of specificity for immune systems. By concentrating TAAs or antigenic peptides with optimized sequences and improved specificity on


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tumor cells, the application of adjuvants, such as the immune enhancers GM-CSF and AS04 for Provenge and Cervarix, respectively, has facilitated exploitation of injective vaccines against various cancers, with pronounced success to increase the 2-5 year survival rate. DNA vaccines have been explored, such as Allovectin-7, which alleviates melanoma. A common side effect of injective vaccines is inflammation and stiffening at the injection site. This deficiency can be avoided using oral vaccination, which is designed on the basis of the active absorption of vaccines or antigens by mucosa immune cells. The packaging of antigens for oral vaccine systems can be optimized using adjuvants that preferentially target intestinal immune cells for presentation. The fact that intestinal mucosa vaccination can boost robust systemic and local immunity in a short period may bring new hope for tumor patients. Apart from adjuvants, current issues with oral vaccines include the efficiency and specificity of the antigens against refractory disease, such as HIV and cancers, with high frequencies of mutation. For cancers, oral vaccines based on multiple antigens are more efficient than those with one antigen. However, since many TAAs are also expressed by normal cells, although at lower levels, self-immunity toward normal tissues might be elicited. The characterization of new and highly specific antigens has become a new research focus in addition to the investigation of adjuvants to achieve enhanced strength and persistence of the immune response. Oral immunization, with minimal injury to tissues and wide acceptability for all ages, has more advantages than traditional injection vaccines. Since the intestinal tract has a large number of immune cells, strengthening the protection, targeting ability, and controlled antigen release of compatible adjuvants plays a decisive role in exploiting oral vaccines. Aiming to establish a strong oral immunization system, synthetic adjuvants with multi-target locations are expected to boost the multi-level immune responses against a variety of diseases and provide more efficient


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protection to the body; therefore, they are promising for oral immunization, protecting human health from disease threats and ensuring social and economic progress, leading to a bright future and improved quality of life. 7. Conclusion Vaccinations prevent many infectious diseases and some noninfectious, chronic, inflammatory and autoimmune diseases. Oral vaccines can elicit robust immune responses by escaping the harsh conditions of the GI tract and stimulating the immune-related tissues in either the small or large intestine. However, few oral vaccine products are available for the prophylaxis of infectious diseases. Adjuvants are molecules, compounds or macromolecular complexes that facilitate a specific immune response to antigens, while causing minimal toxicity or long-lasting immune effects. Modern techniques enable the synthesis of adjuvants for oral vaccination. As integrative vehicles and immunostimulants, synthetic adjuvants can be rationally designed with diverse structures that combine versatile properties, such as intelligent release and targeting and the ability to load and deliver multiple effectors. Therefore, the adjuvants for oral vaccines can stabilize the antigens, modulate the balance between the maintenance and release of the antigens, stimulate cellular uptake, enhance immunity, target the antigens to the desired destination, and respond to environmental stimulation. These advantages show promise for overcoming the harsh GI conditions and barriers for successful exploration of oral vaccines, endowing them with significance for future application.


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FIGURES

Figure 1. Strategy for adjuvanted oral vaccinations targeting M-cells. The micro- or nano-oral vaccine particles comprising adjuvants and antigens, keeping stable and integral in stomach, is transported to immune cells by the microfold cells (M cells) in the small intestine, which locate besides the enterocytes.


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Figure 2. Formation of PCPP based hydrogel. Reversible cross-linking ability of PCPP generated pH sensitive encapsulation and releasing of the antigens. Reproduced with permission from ref 61. Copyright 1989 American Chemical Society.


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Figure 3. Synthesis of dendrimer PAMAM as a candidate for oral vaccination. Derived from from divergent synthesis, the Generation N can perform as adjuvant, with the Generation 6 being most preferred for developing efficient immune response.


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Figure 4. SBA-15 silica nanoparticles as carrier adjuvants. The hexagonal mesoporous SBA-15 silica microparticles can be synthesized with PEO-PPO-PEO as organic structure-directing agents, and further utilized as antigen delivery vehicles for oral vaccination.


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Figure 5. M cell as main gateway for mucosal vaccines. In mucosal vaccination, antigens transferred along M cells are loaded by DCs, and migrated to T-cell areas. T-cells are activated to induce recombination and somatic hypermutation in B cell follicle. Then the antigen-specific IgA can be converted into SIgA in the lumen. Reproduced with permission from ref 114. Copyright 2014 Nature Publishing Group.


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Figure 6．pH sensitive system for large intestine target vaccination. Modified with pH sensitive covering FS30D, PLGA nanoparticles encapsulating PCLUS3-18IIIB (CD4+ T cell helper epitopes fused with HIV Env CD8+ CTL epitope) and TLR ligands (MALP2+poly(I:C)+CpG) can serve as effective vaccine delivery system when they are deposited in large intestinal lumen.


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Figure 7． Schematic illustration of synthesis and controlled release property of the pH and trypsin-responsive bilayer (PTRBL) PMMMA-PLGA based oral vaccine system. The pHdependent phase-transition performance leads to large intestine targeted release through small intestine escaping mechanism owing to the surface negative charge repulsion. Reproduced with permission from ref 99. Copyright 2016 Elsevier.


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Table 1. Synthetic Adjuvants with Versatile Properties and Functions for Optimizing Oral Vaccination adjuvant

sample

properties

study design

PLA

PEG-PLA

Phagocytizable by immune cells PLA particles entrapping rotavirus were 41, 42 administered orally to mice.41 PLGA NPs loading H. pylori lysates were orally Enhanced cell uptake by APC; administered to mice. able to protect the antigens 32, 44, 45 PLGA particles entrapping BSA were delivered to mice orally. HBsAg was encapsulated and orally More stable administered in mice model.

PAS

Porous; continuous fast antigen release 58, 59

TS-PDMS

Biocompatible; able to entrap 5- TS-PDMS-grafted MPs entrapping HSA 6% w/w protein were orally-administered to mice.

PCPP

Biocompatible, pH responsive

PCEP

immunization outcome

organic polymers linear polymers

PLGA

long-lasting IgG and IgA antibody tilters 41 Mucosal and systemic immune responses 51- 53 Higher serum IgG level; similar IgG2a/IgG1 ratio 54 Humoral, mucosal (sIgA), cellular (TH1) immune responses; IL-2 and IFN responses 56

cross-linkable materials

starch-based MPs

cross-linkable Polyphosphazene

61,

62

polymeric hydrogels

PMA

branched polymers

SB(43)-PVALg-PLGA

Carboxylate functionalities; hydrolytically degradable 63 Biocompatible; degradable; environment sensitive Surface charged; with antigen affiliation capability

TGDK dendrimers

Able to target M cells

Micelles

Phosphatidylcholine

Encapsulate BacHA(antigen) in reverse micelle

lipid vehicles

Liposomes (Lip)

Antigen-protective, stimulate endocytosis by M cells 84-86, 88

Inactivated rotavirus in PAS was administered to mice orally and intramuscularly. HSA coupled to the PAS MPs was administered to mice orally by gastric intubation.

Significant antibody levels and neutralizing effect.58 Diversified immune response, especially strong mucosal response (IgA) 59 Antibody secretion in PP; lymphocyte reactivity; enhanced spontaneous Th2-cytokine numbers.60

Polymer–antigen (inactivated H1N1 and H3N2) were parenterally administered to mice.62 PCEP with influenza X:31 antigen were codelivered through oral routes in mice.64 PMA loading Cr-EDTA acid were administered orally to sheep. SB(43)-PVAL-g-PLGA adsorbed TT was given to mice by orally, nasally and intraperitoneally TGDK conjugating Rhesus cyclopeptide was orally-administrated to rhesus macaque.

HAI antibody titers; IgG1 and IgG2a isotypes 62

En-BacHA was delivered to the GI tract of mice.

Serum IgG and mucosal IgA responses; strong immune protection 82

IgA, IgG1 and IgG2a titers 64 The hydrogels bypassed the first stomach and released the antigens in the lower GI tract 66 IgG and IgA serum titers 68 Stool IgA response; neutralizing activity against SIV infection 75

self-assembled Adjuvants

LiposoMAs DSPC/cholesterol functionalized Lip DOTAP Lip with tremellaalginate bilayers Orasomes Bilosomes GM-bilosomes charged polymers

PDDA, PEI, CTS TM65CM50CS PMMMA

Lip loaded S. mutans carbohydrate or ciprofloxacin was orally-administered to humans.84, 86 Lip-encapsulated influenza DNA vaccine was given to mice orally.85 Decorated with mannose OVA was loaded in LiposoMAs and administered derivative and lipid A; safe to mice via oral mucosa. Increased uniformity of the NPs, Lip-encapsulated-JE Virus Ns1 was orally low leakage rate administered to mice. Able to self-close; positively The liposome-encapsulated ND vaccine was charged; nanosized administered orally to chicks. pH sensitive; resistant to an alginate–tremella–lips encapsulated H5N3 acidic pH was administered to mice through oral route. Homogeneous; stable; able A mouse gut loop model was used. to adhere FAE from PP Bac-VP1 associated with bilosomes were 10, 93 Antigen-protective administered orally to mice.10 Stable with sustain antigen GM-bilosomes/BSA were administered orally to release mice. Electropositive; able to promote 3DC/BSA coated with PDDA, CTS and PEI was adsorption administered to mice orally or intramuscularly. Positively charged; swelling Ovalbumin in TM65CM50CS-coated Ca-alginateability; mucoadhesive yam MPs was orally administered to mice. pH responsive; stable under the PMMMA encapsulating the PLGA/TRX-SIP acid conditions NPs were administered orally to tilapia.

Mucosal or IgA response 84- 86 Mucosal immunoresponses; Th1/Th2 immune response; cellular/humoral immunity 87 Serum IgG response; elevated expression of IL-1β, IL-6 and TNF-α in AmB-treated mice.89 Increased antibody titre and immune protection than the marketed La Sota® vaccine 90 Intestinal s-IgA; opening of tight junctions of the Caco-2 cell monolayer.91 Lectins may be used to achieve M cell targeting of oral delivery vehicles.92 Higher IgG and IgA responses compared to non-associated Bac-VP1 10 Mucosal immune response, cell mediated immune response 95 delayed peak titer of IgG and elevated IgA titers showing mid-to-late immune responses 97 IgG and IgA titers 98 IgM response; durative immune protection on tilapia against GBS disease 99

inorganic Adjuvants Silica based particles

SBA-15 S1 and S2

Carbon based particles C1 CaCO3

Stable; biocompatible; mesoporous; sustained release Stable; biocompatible; porous for sustained release Antigens are protected and transported to the M cells

SBA-15/BSA was orally-administered to high and low antibody responsive mice. BSA loaded in S1 (430 nm), S2 (130 nm) and SBA-15 were administered to mice. C1/BSA and administered to mice orally.

High level of IgG and IgA titers, Th1 and Th2 mediated responses 107 High level of IgG titer, mucosal IgA, Th1 and Th2 mediated responses 111

Stabilize the antigens

Self-adjuvanting OVA-KFE8/CaCO3 composite micro-particles were administered orally to mice.

Mucosal IgA responses 112

Effective IgG1 and the IgG2a response 105


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the National Nature Science Foundation of China (31300650, 31270790), the Key Project of Science and Technology of Fujian Province (2013N0039, 2012Y0070, and 2013Y0082), National Thousand Talents Program of China, and Scientific Research Starting Foundation for Returned Overseas Chinese Scholars of State Human Resource Ministry of China for support on this work.

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Table of Contents Graphic and Synopsis


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Chemical Synthesis, Versatile Structures and Functions of Tailorable

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