key: cord-0990167-7t1bzc7f
authors: Barclay, T.; Petrovsky, N.
title: Vaccine Adjuvant Nanotechnologies
date: 2016-10-07
journal: Micro and Nanotechnology in Vaccine Development
DOI: 10.1016/b978-0-323-39981-4.00007-5
sha: 71491672e3ebbdb760264a8a3318e5207a7456e6
doc_id: 990167
cord_uid: 7t1bzc7f

The increasing sophistication of vaccine adjuvant design has been driven by improved understanding of the importance of nanoscale features of adjuvants to their immunological function. Newly available advanced nanomanufacturing techniques now allow very precise control of adjuvant particle size, shape, texture, and surface chemistry. Novel adjuvant concepts include self-assembling particles and targeted immune delivery. These individual concepts can be combined to create a single integrated vaccine nanoparticle-combining antigen, adjuvants, and DC-targeting elements. In the process, the concept of an adjuvant has broadened to include not only immune-stimulatory substances but also any design features that enhance the immune response against the relevant vaccine antigen. The modern definition of an adjuvant includes not only classical immune stimulators but also any aspects of particle size, shape, and surface chemistry that enhance vaccine immunogenicity. It even includes purely physical processes such as texturing of particle surfaces to maximize immunogenicity. Looking forward, adjuvants will increasingly be seen not as separate add-on items but as wholly integrated elements of a complete vaccine delivery package. Hence, vaccine systems will increasingly approach the complexity and sophistication of pathogens themselves, incorporating highly specific particle properties, contents, and behaviors, all designed to maximize immune system recognition and drive the immune response in the specific direction that affords maximal protection.

Reduced immunogenicity and thereby efficacy is an unfortunate downside to vaccines based on highly purified or synthetic antigens. This requires the use of vaccine adjuvants to improve vaccine immunogenicity. The term adjuvant has traditionally referred to as any substance that when added to a vaccine antigen increases its immunogenicity. 1 As such, the study of adjuvant action is made complicated by the wide diversity of agents including small molecule immune modulators, mineral salts, oil emulsions, and even whole viruses or bacteria. These agents all exhibit adjuvant action in vivo with no one common feature explaining this activity. Almost certainly, these compounds work through many and varied different mechanisms to enhance immune responses to coadministered antigens. Increasing use of small protein or peptide antigens allows greater control over vaccine design and synthesis but has further exacerbated issues of poor immunogenicity. This necessitates closer examination of how best to design vaccine delivery and adjuvant systems to maximize antigen immunogenicity, assist targeted immune delivery, and provide potent adjuvant activity. Most importantly, this must all be done in a manner that does not compromise vaccine tolerability or safety. 2, 3 The immune system has evolved to protect against invading pathogens. As pathogens present as nano-to microsized particles, it should not be surprising that the immune system has evolved strategies to specifically recognize and respond to particles of particular sizes and shapes. 4, 5 This helps explain the boost to immunogenicity when antigens are presented to the immune system as virus-like particles (VLPs) rather than as soluble proteins. Particle size was shown to be important for uptake by dendritic cells (DCs), with spherical particles less than 500 nm in diameter having better uptake than larger particles. 6 Size is also relevant to T cell maturation whereby poly(lactide-co-glycolide) (PLGA) microparticles modified with recognition and costimulatory ligands with similar dimensions to DCs were better able to induce T cell activation when compared with nanosized particles 7 that were rapidly phagocytosed. 8 While the idea that size is important to vaccine action is now well accepted, it is also likely that shape, texture, and surface chemistry are equally highly relevant to adjuvant action, 8 albeit less well-explored.

Shape is known to be important to particle uptake in vivo, half-life, and biodistribution. 9, 10 For example, cylindrical silicon particles exhibited strong liver accumulation, whereas discoidal particles accumulated away from the liver and spherical particles were distributed evenly across tissues. 11 Decreasing the diameter of spherical particles from 3 to 0.7 µm resulted in increased accumulation in reticuloendothelial tissues. 11 These tissues are rich in various immune cells, and consequently accumulation in the reticuloendothelial system is an advantage from a vaccine standpoint. Notably, high-axial-ratio nanoparticles were shown to better target draining lymph nodes and thereby enhanced antigen presentation and memory B cell responses. 12, 13 Surface chemistry has also been shown to be important for adjuvant particle properties, with uptake of larger particles by DCs being enhanced by a positive surface charge. 6 Furthermore, while 25 nm diameter polyhydroxylated and polymethoxylated poly(propylene sulfide) nanoparticles and polystyrene nanoparticles were all equally efficient in inducing DC uptake, only the polyhydroxylated particles induced high levels of DC maturation, 14 an effect attributed to the polyhydroxylated surface activating complement thereby acting as an immune danger signal for DC activation. 14 Use of hydrophilic polymeric coatings such as polyethylene glycol and polyzwitterions with an overall neutral surface charge that bind water to the surface has also been shown to change particle behavior, 8 with the hydrophobic regions of amphiphilic systems considered to act as immune danger signals that provide adjuvant effects. 15 Mechanical properties are yet another factor that need to be taken into account when assessing the immune effect of particles. Macrophages and DC have been shown to preferentially phagocytose rigid particles even if these have identical surface chemistry to softer particles. 16 Hence making particles more flexible so that they mimic red blood cell properties was shown to increase their half-life in vivo, 11, 17, 18 whereas making them more rigid should improve their uptake by DCs and thereby their immune action.

Increasingly sophisticated particle manufacturing approaches have enabled ever-tighter control of the size, shape, external chemistry, and mechanical properties of adjuvant particles. This has provided unique opportunities to modify vaccine and adjuvant particles with the aim to maximize their biological action. The remainder of this chapter will describe a wide variety of nano-and microparticulate vaccine and adjuvant systems, their method of manufacture, and how size, shape, and surface chemistry are all important to adjuvant action ( Fig. 7.1 ). 

Emulsions are composed of a dispersion of droplets of one liquid in another immiscible liquid, commonly described as either water-in-oil or oil-in-water emulsions. 21 In use as adjuvants, the droplets in an emulsion act as particles and are perceived by the immune system as such. There are two different forms of nanoscale emulsions, both of which are defined as having droplet radii from 1 to 100 nm, which is sufficiently small to generate transparent, isotropic liquid media. The first form of nanoscale emulsion has historically been termed a microemulsion but is better described as a nanodimensional lyotropic liquid crystalline phase. 22 In a microemulsion the structural organization of the two liquids is supported by a relatively large concentration of surfactant. Formation is by spontaneous selfassembly with relatively gentle mixing, resulting in stable organization at thermodynamic equilibrium. 22, 23 By contrast, what is termed a nanoemulsion generally uses significantly less surfactant and hence requires high shear forces to make a metastable structure. 22, 24 Emulsion adjuvants are thought to work through a depot effect, creating inflammation around the site of antigen injection and thereby enhancing humoral immune responses. 25 Early examples of emulsion adjuvants were water-in-mineral-oil emulsions. 26, 27 However, such adjuvants were problematic as mineral oil is not biodegradable, is proinflammatory, and has long-term persistence in the body, thereby leading to major inflammatory side effects including injection site granulomas, pyrexia, and long-term safety issues. 28 Care must also be taken with any emulsion adjuvant to avoid any oxidation of the oil component as this could otherwise lead to increases in vaccine reactogenicity. Generally, oil-in-water nanoemulsions using biodegradable oils have a better tolerability profile than water-in-oil formulations. A well-known example of an oil-inwater nanoemulsion adjuvant is MF59, which is composed of squalene oil in a citrate buffer using nonionic surfactants. 29, 30 The nanoemulsion is key to the adjuvant effect of MF59 as when tested individually the various components of MF59 failed to demonstrate adjuvant activity. 31 MF59-based vaccine formulations have been used extensively in research, 32 and recent work has included tests as a cationic nanoemulsion delivery system for delivery of a self-amplifying mRNA vaccine. 33 Nonetheless, the bulk of research has been for influenza vaccines, 34 and while MF59 is licensed for this purpose in elderly humans 31 and has extensive safety data in the elderly population, more limited safety data are available for use in children. Of potential relevance, a 2009 pandemic influenza vaccine containing AS03, a squalene-based nanoemulsion that is similar to MF59 except for the addition of tocopherol, was associated with a rise in childhood cases of narcolepsy, 35, 36 an autoimmune sleep disorder. This has put the question of pediatric safety of squalene-based emulsion adjuvants back under the spotlight.

The immunogenicity of emulsion adjuvants can be increased further by coformulation with additional immune-stimulatory components. Interestingly, the original MF59 squalene emulsion adjuvant was initially designed as a delivery system for muramyl tripeptide phosphatidylethanolamine (MTP-PE), a modified mycobacterial peptidoglycan with potent adjuvant activity. 37 MTP-PE activates innate immune receptors including nucleotide-binding oligomerization domain-containing protein 2 (NOD2) 38 and various Toll-like receptors (TLR), 39 leading to nuclear factor kappa-B (NFκB) activation and induction of inflammatory cytokines. 40 However, the MTP-PE component was ultimately removed from MF59 because of excessive reactogenicity. 41 A more recent version of a combined adjuvant emulsion and immunostimulator approach is the AS02 adjuvant, which is an oil-in-water emulsion combined with QS21 saponin and the TLR4 agonist, monophosphoryl lipid A (MPL). 42 Intranasal delivery of vaccines provides advantages in terms of convenience and induction of mucosal immunity, a major benefit when protecting against respiratory or mucosal pathogens. 43, 44 A nasal vaccine containing a soybean oil nanoemulsion adjuvant successfully enhanced humoral and cellular responses in mice, [43] [44] [45] [46] [47] consistent with the utility of this approach. A library of more than 100 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions containing various cationic and nonionic surfactants were tested in mice and demonstrated that varying the physicochemical properties of the surfactant components (charge, surfactant polar head size, and hydrophobicity) and the surfactant blend ratio of the intranasal adjuvant formulations could modulate the strength and type of the immune response. 48 This indicates that there may be considerable scope for further optimizing nanoemulsion adjuvants for intranasal use. However, the tolerability and safety in humans of this approach have yet to be confirmed. It was recently shown that nanoemulsion adjuvants induce epithelial cell death via activation of caspases 1, 3, and 6-9. 49 Furthermore, the safety of nasal vaccine adjuvants has remained under a cloud since the experience of NasalFlu, an intranasal influenza vaccine containing a detoxified enterotoxin adjuvant, that was associated with increased cases of facial nerve palsy in clinical trials. 44 Multiple or double emulsions, most often water-in-oil-in-water formulations, have also been used in vaccine research. 50 However, water-in-oil-in-water multiple emulsion proved to be less stable and induced an excessive inflammatory response when compared with an oil-in-water microemulsion of isopropyl myristate, which successfully enhanced humoral responses to rabies vaccine in mice. 51 These stability issues arise as there are two interfaces with high free energy to stabilize, generally requiring two surfactants that may interact, thereby interfering with stabilization. Also, for multiple emulsions there is the potential for an osmotic pressure to develop, placing further strain on the system. 52 Even simple emulsions are not without issues. Nanoemulsions not at thermodynamic equilibrium have poor stability, and microemulsions suffer from the need for high concentrations of surfactants, which may cause toxicity in vivo. Furthermore, the stability of any microemulsion can be easily compromised by dilution, heating, or pH changes.

Various nanoparticles constructed from disordered, precipitated polymers, also described as nanobeads, have been used for vaccine delivery. Poly-ε-caprolactone nanoparticles of two sizes were made, with mean diameters of 61 and 467 nm, by solvent evaporation. Only the smaller diameter particles were found to have an adjuvant effect, eliciting both cellular and humoral responses in mice. 53 Polystyrene nanobeads covalently conjugated to ovalbumin were found to best activate CD8 T cells when they were 40-49 nm diameter when beads ranging in size from 20 to 2000 nm diameter were compared. 54, 55 Another study found that larger beads with diameters from 93 to 123 nm were best at activating CD4 T cells 55 Thus, it may be possible to specifically tune nanoparticle size to induce either cellular or humoral immunity. In another system poly(γ-glutamic acid) grafted with l-phenylalanine ethyl ester on 53% of the carboxylic acid groups self-assembled upon the addition of water from organic solution into nanoparticles with diameters of 150-200 nm. 56 In the presence of ovalbumin the nanoparticles encapsulated the antigen and targeted it to DCs, resulting in enhanced DC maturation and immunity. 56,57

Inorganic nanoparticles have been used as vaccine delivery vehicles; aluminum oxide nanoparticles having distinctly better performance compared with other metal oxide nanoparticles. 58 Such aluminum oxide nanoparticles of 113-355 nm diameter elicited adjuvant effect when peptomers derived from an HIV antigenic sequence and retaining the native α-helical conformation were covalently attached to the external surface. 59, 60 Aluminum oxide nanoparticles were also functionalized with ovalbumin and shown to enhance DC activation of T cells via an autophagy-dependent mechanism. 58 Particle size was also shown to be important as while both 60 and 200 nm nanoparticles were effective in vitro, the 60 nm aluminum oxide particles were much more effective in being transported to the draining lymph nodes in vivo.

VLPs can be made using recombinant protein techniques whereby antigenic proteins such as HBsAg are synthesized in yeast, following which the antigen self-assembles into 22 nm VLPs. 61 VLPs are safe and immunogenic, making them ideal vaccine antigens. 62, 63 However, cell-culture systems used for manufacture of VLPs can be low yielding and expensive, and hence synthetic methods have been developed for VLP production. For example, short linear synthetic proteins with coiled-coil domains were used for self-assembly of α-helical motif-containing nanoparticles able to present repetitive epitopes on their surface. 64, 65 These synthetic particles had a 25 nm diameter and elicited strong immune responses to surfaceexpressed epitopes. [64] [65] [66] A related nanoparticulate vaccine delivery system was also assembled from peptide chains containing a coiled-coil sequence to drive helical assembly. In this instance the peptide was modified at the N-terminus with a dual chain lipid and with synthetic epitopes at the C-terminus. Upon dispersion in aqueous solution, the construct aggregated into ordered synthetic particles having a diameter of 17-20 nm and presenting multiple copies of the antigen on the surface that induced a humoral immune response. 67 

In another synthetic system polypeptides designed to self-assemble into β-sheet-rich nanofibers with epitopes attached to the C-terminus generated strong antibody responses without inflammation. 68 Similarly to VLP's, the self-assembled structure was critical to the adjuvant affect, the unassembled sequence having no adjuvant properties. 69 It has been shown that the nanofibers are internalized by DCs and macrophages at the injection site, resulting in increased expression of the activation markers CD80 and CD86 and enhanced production of T follicular helper cells and germinal center B cells. 68 Nanostructuring of materials within tissue depots may also generate adjuvant activity. Peptides having alternating hydrophobic and hydrophilic amino acids can undergo a sol-gel transition upon exposure to physiological conditions whereby they form a hydrogel made up of interconnected nanofibers. 70 In this way a mixture of biphasic oligopeptide, antigen, and adjuvants can be injected with the peptides, subsequently undergoing postinjection self-assembly into hydrated nanofiber gel matrices, forming an antigen and adjuvant depot in the aqueous phase. 71 A self-assembling nanofibrous hydrogel induced an antibody response when tested as a vaccine delivery platform, either alone or formulated with CpG adjuvant (TLR9 agonist) as a delivery system for recombinant hepatitis B surface antigen (HBsAg). 71 Similarly, the self-assembling peptide Q11 conjugated to a CD8 T cell epitope of ovalbumin elicited a strong antigen-specific CD8 T-cell response. 72

Liposome-based vaccines were an early form of particulate adjuvant formulation based on the use of cell-like spherical lipid bilayer vesicles containing antigens within the internal aqueous compartment, within the lipid bilayer, or attached externally. 73, 74 For example, 165 nm-diameter liposomes assembled from cationic lipid, cationic polymer, and plasmid DNA were shown to target antigen to draining lymph nodes, resulting in enhanced DC activation and immunity. 75 Coating of these liposomes with a cholesterol-modified mannan further enhanced the therapeutic anticancer action of an oncoprotein vaccine. 76 As liposomes themselves are generally poorly immunogenic, they may require combination with other immunostimulatory adjuvants to enhance their immunogenicity. 4 For example, liposomal nanoparticles self-assembled from a mixture of phospholipids containing 5% pegylated lipid to prevent protein binding were used to target cyclic diguanylate as an agonist of stimulation of INF genes to draining lymph nodes, thereby enhancing vaccine potency. 77 Single bilayer liposomes have also been constructed using viral lipid envelopes, retaining the viral cell binding and fusion glycoproteins and resulting in 150 nm-diameter virosomes that promote attachment to respiratory mucosal surface and subsequent fusion release of contents into the cytoplasm of endocytosing cells. 78 Further, the viral components enhance the efficiency of the interaction with antigen-presenting cells (APCs), making them an ideal vaccine delivery system. 79, 80 More recent virosome research has included the incorporation of additional components to improve the particle formation and stability characteristics as well as enhance immunogenicity. 81 For example, an amphiphilic saponin derivative (GPI-0100) was incorporated into a virosome and provided potent immunogenicity to allow the use of very low antigen doses. 82

Electrostatic interactions are an important part of nanoparticle vaccine formulations, both between components of the formulation 83, 84 and between the formulation and the anionic cell membranes of APCs. 85 These charged interactions have demonstrated importance in various nanovaccines, including liposomal systems, 86 but perhaps are best exploited in polymeric systems. A mixture of an anionic poly(γ-glutamic acid) acid grafted with phenylalanine ethylester and a cationic ε-polylysine was self-assembled from aqueous solution into nanoparticles with 200-300 nm diameter. 83 When formed in the presence of ovalbumin, the particles were loaded with antigen and induced effective cellular and humoral immune responses in immunized mice. 87 Poly(γ-glutamic acid) has also been eletrostatically attached to complexes of dendrigraft poly-l-lysine with plasmid DNA. In this instance the poly(γ-glutamic acid) moderates the cytotoxicity of the electrostatic complex to enable delivery and expression of genes in the spleen, suggesting application in vaccine formulations. 88 This is related to a similar gene delivery formulation in which electrostatic interactions between positively charged polyethyleneimine and negatively charged plasmid DNA and poly(γ-glutamic acid) were used to construct a nanoparticle DNA vaccine. This formulation conferred protection against malaria infection in mouse models. 84 Standard PLGA particles have negative charge, and electrostatic interactions have been identified as a critical aspect of the binding to protein antigens. Consequently, protein binding can be tuned through adjusting the pH of binding conditions to optimize the charge differential between components. 89 PLGA nanoparticles have also been constructed with positive charge using PRINT technology through the incorporation of cationic additives. These particles were specifically designed to electrostatically interact with commercial hemagglutinin antigens to generate an influenza vaccine with enhanced immune responses compared with the hemagglutinin alone. 12,13

Lipid-like amphiphiles have been used in several self-assembling nanoparticulate-based self-adjuvanting antigen delivery systems because their structure mimics bacterial lipoproteins and as such can activate the immune system. 90 This type of vaccine formulation includes a hydrophobic core covalently attached to a hydrophilic peptide antigen such that the antigen epitope is presented on the surface of the nanoparticle. 91 A nontoxic, dendritic poly tert-butyl acrylate polymer core has been used and when conjugated to relevant peptide epitopes induced cellular responses able to kill human papillomavirus-infected cancer cells. 92 Epitope packing on the particle surface was dense enough to maintain a native conformation and enable proper recognition by the immune system. 91, 92 In other examples the particle core was made up of either lipid-like peptides 93 or lipidated peptides. 94, 95 Such formulations include a peptide epitope from a hookworm protein flanked by coil-producing sequences attached to a lipid-like peptide core. The native alpha helix structure of the epitope presented on the surface of the nanoparticle was able to generate antibodies in mice. 96 

As previously suggested by their use in emulsion and liposome adjuvants, saponins can provide potent immunogenicity to vaccine formulations. A commonly used adjuvant is the saponin QS21, which is an acylated saponin at the 4-hydroxyl position on fucose with two linked 3,5 dihydroxy-6-methyloctanoic acids. 97 QS21 induces inflammatory cytokines and imparts a Th1 bias in vaccine responses, 98,99 but because of its ability to lyse cell membranes, hemolysis and injection site pain are major limiting factors in its use. 100 QS21's toxicity can be reduced by forming it into an immune-stimulating complex (ISCOM), which is a spherical particle of ∼40 nm diameter that self-assembles from specific mixtures of cholesterol, phospholipids, and QS21. [101] [102] [103] An advantage of ISCOMs is that the toxic hemolytic effect of the saponin is moderated by its incorporation into the liposomal structure while its immune-stimulatory properties are retained. The liposomal particle also helps target the saponin adjuvant and antigen formulation to the draining lymph nodes. 5 While initially thought important for immunogenicity that antigens were loaded inside the ISCOM structure, it is now recognized that adjuvant action can be achieved by simple admixture of the antigen with the ISCOMs, a major advantage as many antigens are difficult to load into the particles. 104 

Many plant-based polysaccharides have been shown to have adjuvant activity, with the additional benefit that as sugar-based compounds polysaccharides are generally extremely safe and well tolerated. 20 While some polysaccharides (eg, mannan) may have adjuvant activity when in soluble form, most are only adjuvant active when present as insoluble particles. This is clearly seen with the polysaccharide inulin (β-d- poly(fructo-furanosyl)-d-glucose), a natural plant-derived storage carbohydrate of plants of the Compositae family that has no immunological activity when in the usual soluble form but has potent adjuvant activity when crystallized into the nanostructured delta inulin microparticles. [105] [106] [107] [108] Delta inulin particles have been shown to enhance humoral and cellular immune responses to a wide variety of viral, bacterial, and protozoan antigens as well as toxins and allergens. For example, enhanced vaccine protection was seen in models of influenza, 109, 110 Japanese encephalitis, 111, 112 West Nile virus, 113 hepatitis B, 114 human immunodeficiency virus, 115 anthrax, 116 SARS coronavirus, 117 listeriosis, 118, 119 and African horse sickness. 120 Its adjuvant effects are seen across a broad range of animal species including mice, rats, guinea pigs, rabbits, chickens, dogs, sheep, monkeys, horses, and camels. It has proved effective in human studies when combined with a recombinant pandemic influenza vaccine, 121 a hepatitis B vaccine, 122 or a bee sting allergy vaccine. 123 An interesting feature of delta inulin is its ability to enhance adaptive immune responses even when injected a day prior to the antigen, a feature not shared by alum adjuvant. 114 Delta inulin does not work like other polysaccharide adjuvants, as it has not been found to activate innate immune receptors such as TLRs, Dectin-1, or the inflammasome. Instead, delta inulin adjuvant appears to work by directly modulating DC function, thereby resulting in enhanced antigen presentation to memory T and B cells. This was recently shown in human subjects to translate into enhanced B cell receptor affinity maturation with upregulation of activation-induced cytidine deamidase in day 7 postimmunized plasmablasts. 124 Notably, delta inulin has proved safe and effective when administered to pregnant dams 125 or their 7-day-old mouse pups 126 and, in a large animal model, to either pregnant mares or their foals, 127 a feature that may be attributable to its noninflammatory mode of action. Interestingly, a powder particle formulation of delta inulin has recently been shown to be safe and effective when administered with influenza vaccine directly into the lungs of mice, 128 opening up a further novel vaccine application of this technology.

Other polysaccharide particles with adjuvant activity include dextran, β-glucan, lentinan, zymosan, mannan, and chitosan. 20 These all work through the action of specific innate immune receptors expressed on APCs known as lectins that specifically recognize and respond to sugars. These include the β-glucan receptor, the mannan receptor, Dectin 1, and TLR. Activation of these innate immune receptors results in NFκB activation and production of proinflammatory cytokines that enhance adaptive immune responses. Hence delta inulin currently appears to be the only polysaccharide particulate adjuvant that works through an alternative noninflammatory pathway.

Given the high expression of lectins on APCs, decorating the surface of vaccine or adjuvant particles with sugar groups can assist vaccine particle targeting to APCs. For example, mannose has been used to target plasmid DNA-containing liposomes to macrophages. 129 Coating of cationic liposomes with mannan significantly enhanced the ability of a DNA vaccine to induce HIV-specific cellular immunity and also enhanced the activity of a DNA vaccine against melanoma. 130, 131 Mannosylated niosomes composed of span 60, cholesterol, and stearylamine (all coated with the modified polysaccharide O-palmitoyl mannan) have been used as orally administered DNA vaccine carriers with a demonstration of enhanced mucosal immunity in mice. 132 A similar approach using O-palmitoyl mannan coating was used to target niosomes to Langerhan's cells in the skin after topical delivery. 133 Chitin, a linear β-1-4-linked polymer of d-glucosamine and N-acetyl-d-glucosamine extracted from shrimp, and chitosan, obtained by partial deacetylation of chitin, act by binding innate receptors including Dectin-1, macrophage mannose receptor, and TLR-2. 134 Chitosan particles produced by cross-linking with a counter ion were used to entrap antigen and enhance its immunogenicity in mice. 135 By virtue of their mucoadhesive qualities, particles coated with chitin and its derivatives have been extensively used as nasal adjuvants for delivery of inactivated 136 or even live viral vectors. Hence mucosal delivery of adenoviral vectors microencapsulated in a chitosan microparticle not only helped to protect and improve the viability of the viral vector but also made its release dependent on cell surface contact. 137 

Given the ability of specific nanoparticles to target APCs, as discussed previously, such particles can also be loaded with immune stimulators to further increase their adjuvant potency. MDP (N-acetyl muramyl-l-alanine-d-isoglutamine), a mycobacterial peptidoglycan with potent adjuvant activity, 37 binds and activates NOD2 38 and TLR receptors, 39 leading to NFκB activation, inflammatory cytokine production, and DC maturation. A modified form, MTP-PE, was the key immune stimulator for which the original MF59 emulsion adjuvant was designed as a delivery system, given the highly hydrophobic nature of MTP-PE. 40 Ironically, the combined MTP-PE in MF59 formulation was abandoned because of excess reactogenicity, 41 but the MF59 vehicle was found to have some adjuvant activity in its own right, and hence its use in influenza vaccines was continued. In a similar fashion, liposomes formulated with DTP-GDP (N-acetylglucosaminyl-N-acetylmuramyl-l-Ala-d-isoGlu-l-Ala-glyceroldipalmitate) had potent adjuvant activity and induced remission in human metastatic colorectal cancer, although reactogenicity with fever, chills, and hypotension was a problem at higher doses. 138, 139 The combination of chitosan microparticles with the mucosal toxin-based adjuvant LTK63 significantly enhanced the immunogenicity of an intranasal group C meningococcal polysaccharide vaccine in mice. 136 Similarly, intranasal administration of alginate-coated chitosan nanoparticles loaded with antigen and CpG adjuvant enhanced antibody and cellular responses in mice. 140 CpG has also been incorporated as the hydrophilic head group into a lipid-like adjuvant macromolecule, the hydrophobic tail composed of cholesterol, simple monoacyl, or diacyl lipidic groups. This formulation was used to successfully target CpG adjuvant and the antigen to lymph nodes, thereby helping decrease systemic CpG toxicity while enhancing vaccine immunogenicity. 141 

Emulsions are not only useful as adjuvants, as discussed earlier, but they can also be used as templates to produce controlled-size solid adjuvant particles (Fig. 7.2 ). Using this method, the polysaccharide inulin was cosolubilized with an ovalbumin antigen into a water-in-oil emulsion with the inulin then precipitated by the slow addition of acetone. This resulted in formation of spherical 1.5 µm-diameter inulin particles encapsulating the ovalbumin with high efficiency. 142 These antigen-loaded inulin microparticles improved APC uptake of ovalbumin and enhanced antiovalbumin antibody responses in mice. Emulsions have similarly been used as templates to precipitate many other biodegradable polymeric nanoparticles for use in vaccine delivery, including the formation of PLGA nanoparticles from a nanoemulsion. The PLGA nanoparticles acted as an antigen delivery system although, unlike inulin particles, the PLGA particles themselves appeared to have no intrinsic adjuvant properties with an immunomodulator being required to create a strong immune response. 15, 143 By contrast, poly(γ-glutamic acid)-based nanoparticles generated an immune response without the requirement of other adjuvants. 57 Emulsion polymerization is another method whereby polymeric particles of size defined by the emulsion can be generated. 144 In this method the soluble polymer droplets within the emulsion are chemically cross-linked such that the droplet-sized particles remain solid when the emulsion is removed. Poly(ortho ester) microparticles of 5 µm diameter produced in this way were used to deliver plasmid DNA antigen to APCs. The acidic conditions in the APC lysosome degraded the orthoester bonds holding the particles together, thereby releasing the DNA. The DNA was then transcribed into protein that was presented to T and B cells, resulting in a cellular and humoral memory response. 144 In another example, size-tuneable cross-linked poly(propylene sulfide) nanoparticles with sizes ranging from 25 to 250 nm were prepared from emulsions, with monomers in the water phase stabilized by Pluronic F-127. 145 Particle size was then controlled by varying the concentrations of the monomer and surfactants in the emulsion. These particles presented pluronic hydroxyl groups at the surface, resulting in complement activation, DC maturation, and a strong adjuvant effect. 14 Such particles are unstable and release any attached drug under oxidative conditions, such as those found in the lysosome. 14, 145 The ability to produce particles of different sizes enabled the effect of particle size on antigen delivery to be directly studied, revealing that small 20-25 nm nanoparticles delivered attached antigen to draining lymph nodes with 10-fold higher efficiency than 100 nm particles. 14, 19 Other studies using these particles have shown that particle surface chemistry is important to the action of nanoadjuvants with, for example, polyhydroxylated surfaces performing much better than polymethoxylated surfaces 14 despite both particles being targeted to the DCs.

Controlled production of defined-size adjuvant nanoparticles can also be achieved through particle replication in nonwetting template (PRINT) technology. 9,13 PRINT techniques use photolithography to create a template that is then transferred to a perfluoropolyether elastomer mold. The mold is filled with the desired material under pressure and then allowed to set. Release of the material from the mold produces particles in the nano-or microscale that can have complex and highly conserved morphology. 9 This technology has been used for a range of medical applications, including production of stimuli-responsive targeted drug delivery vehicles 9,146 and vaccine adjuvants. 13 The size, shape, and chemistry of PRINT nanoparticles can be optimized to maximize their transport to lymph nodes, enabling their use as an antigen delivery vehicle. 12 This work found that anionic 80 x 180 nm cylinders covalently bound to ovalbumin through a 500-dalton polyethylene glycol spacer optimized delivery of antigen to lymph nodes and enhanced vaccine responses. 12 

This chapter highlighted the increasing sophistication of vaccine adjuvant design driven by the convergence of improved immunological understanding of the importance of nanofeatures to adjuvant function together with advanced nanomanufacturing techniques that allow very precise control of particle size, shape, texture, and surface chemistry. Principles from antigen design (eg, particle self-assembly) are increasingly being incorporated into adjuvant nanoparticle design. These individual concepts are now being applied to the design of a single integrated vaccine particle, incorporating antigen, adjuvant, and APC-targeting strategies. In the process, the concept of what is an adjuvant needs to be broadened to not only include specific immune-stimulatory substances but also to include any vaccine design features that enhance immune responses against a relevant antigen. Thus, the definition of an adjuvant could potentially now include aspects of antigen formulation such as nanoparticle incorporation; aspects of particle size, shape, and surface chemistry that enhance immunogenicity; or even purely physical processes such as texturing of the particle surface to maximize immunogenicity. Looking forward, adjuvants will increasingly not be seen as separate add-on items but as wholly integrated elements of a complete vaccine delivery package. Vaccine systems are steadily approaching the complexity and sophistication of the pathogens they mimic, with particle properties and behaviors designed to maximize long-term immune protection while maintaining a high safety profile.

Microparticulate Polysaccharide Adjuvants 135

Nanoparticle Manufacturing Methods 138 7.6.1 Emulsions for template-based particle assembly 138 7.6.2 PRINT-based adjuvant production

Vaccine adjuvants: current state and future trends

New-age vaccine adjuvants: friend or foe?

Freeing vaccine adjuvants from dangerous immunological dogma

Particulate systems as adjuvants and carriers for peptide and protein antigens

Advances in vaccine adjuvants

Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model

A comprehensive platform for ex vivo T-cell expansion based on biodegradable polymeric artificial antigen-presenting cells

Engineering nano-and microparticles to tune immunity

PRINT: a novel platform toward shape and size specific nanoparticle theranostics

Nanofabricated particles for engineered drug therapies: a preliminary biodistribution study of PRINT (TM) nanoparticles

Size and shape effects in the biodistribution of intravascularly injected particles

Rapid and persistent delivery of antigen by lymph node targeting PRINT nanoparticle vaccine carrier to promote humoral immunity

Development of a nanoparticle-based influenza vaccine using the PRINT® technology

Exploiting lymphatic transport and complement activation in nanoparticle vaccines

Peptide-based subunit nanovaccines

Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform

Red blood cell-mimicking synthetic biomaterial particles

In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles

Carbohydrate-based immune adjuvants

Synthesis of nanomaterials in microemulsions: formation mechanisms and growth control

Nanoemulsions: formation, structure, and physical properties

Preparation of metal nanoparticles in water-in-oil (w/o) microemulsions

Formation and stability of nano-emulsions

Adjuvants designed for veterinary and Hum Vaccin

Sensitization and antibody formation after injection of tubercle bacilli and paraffin oil

The mode of action of mineral-oil emulsion adjuvants on antibody production in mice

Outlining novel cellular adjuvant products for therapeutic vaccines against cancer

Design and evaluation of a safe and potent adjuvant for Hum Vaccin

Clinical and immunologic responses to human immunodeficiency virus (HIV) type 1SF2 gpl20 subunit vaccine combined with MF59 adjuvant with or without muramyl tripeptide dipalmitoyl phosphatidylethanolamine in non-HIV-infected human volunteers

The adjuvant effect of MF59 is due to the oil-in-water emulsion formulation, none of the individual components induce a comparable adjuvant effect

MF59™ as a vaccine adjuvant: a review of safety and immunogenicity

A cationic nanoemulsion for the delivery of next-generation RNA vaccines

MF59 adjuvant: the best insurance against influenza strain diversity

AS03 Adjuvanted AH1N1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in Finland

Increased incidence and clinical picture of childhood narcolepsy following the 2009 H1N1 Pandemic Vaccination Campaign in Finland

Adjuvant activity of 6-0-mycoloyl-Nacetylmuramuyl-L-alanyl-D-isoglutamine

Muramyldipeptide and diaminopimelic acid-containing desmuramylpeptides in combination with chemically synthesized Toll-like receptor agonists synergistically induced production of interleukin-8 in a NOD2-and NODl-dependent manner, respectively in human monocytic cells in culture

Enhancement of TLR-mediated innate immune responses by peptidoglycans through NOD signaling

Systemic cytokine profiles in BALB/c mice immunized with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants

Enhancement of humoral response against human influenza vaccine with the simple submicron oil/water emulsion adjuvant MF59

Evaluation of the immune response to RTS,S/AS01 and RTS,S/AS02 adjuvanted vaccines: randomized, double-blind study in malaria-naive adults

Development of immune response that protects mice from viral pneumonitis after a single intranasal immunization with influenza A virus and nanoemulsion

Efficacy, immunogenicity and stability of a novel intranasal nanoemulsion-adjuvanted influenza vaccine in a murine model

Nanoemulsion nasal adjuvant W805EC induces dendritic cell engulfment of antigen-primed epithelial cells

Formulation and characterization of nanoemulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immunogenicity

Safely and immunogenicity of a novel nanoemulsion mucosal adjuvant W805EC combined with approved seasonal influenza antigens

Formulation, high throughput in vitro screening and in vivo functional characterization of nanoemulsion-based intranasal vaccine adjuvants

Nanoemulsionbased mucosal adjuvant induces apoptosis in human epithelial cells

Use of a sustained-release multiple emulsion to extend the period of radio protection conferred by cysteamine

Evaluation of water-in-oil-in-water multiple emulsion and microemulsion as potential adjuvants for immunization with rabies antigen

Multiple emulsions: technology and applications

Fabrication of nanoadjuvant with poly-ε -caprolactone (PCL) for developing a single-shot vaccine providing prolonged immunity

Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors

Type 1 and 2 immunity following vaccination is influenced by nanoparticle size: formulation of a model vaccine for respiratory syncytial virus

Preparation and characterization of biodegradable nanoparticles based on poly(gamma-glutamic acid) with 1-phenylalanine as a protein carrier

Targeting of antigen to dendritic cells with poly( -glutamic acid) nanoparticles induces antigen-specific humoral and cellular immunity

Alpha-alumina nanoparticles induce efficient autophagy-dependent crosspresentation and potent antitumour response

Peptomer aluminum oxide nanoparticle conjugates as systemic and mucosal vaccine candidates: synthesis and characterization of a conjugate derived from the C4 domain of HIV-1 MNGpl20

Immunization of mice with peptomers covalently coupled to aluminum oxide nanoparticles

Synthesis and assembly of hepatitis B virus surface antigen particles in yeast

Priming of class I-restricted cytotoxic T lymphocytes by vaccination with recombinant protein antigens

Immunization of human HIV-seronegative volunteers with recombinant pl7/ p24:Ty virus-like particles elicits HIV-1 p24-specific cellular and humoral immune responses

Nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine

Protective antibody and CD8+ T-cell responses to the Plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine

A nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria

Synthetic virus-like particles from self-assembling coiled-coil lipopeptides and their use in antigen display to the immune system

The use of self-adjuvanting nanofiber vaccines to elicit high-affinity B cell responses to peptide antigens without inflammation

A self-assembling peptide acting as an immune adjuvant

Controlled release of insulin from self-assembling nanofiber hydrogel, PuraMatrix (TM): application for the subcutaneous injection in rats

Vaccine self-assembling immune matrix is a new delivery platform that enhances immune responses to recombinant HBsAg in mice

Antigenic peptide nanofibers elicit adjuvant-free CD8(+) T cell responses

Liposomes as immunological adjuvants

Immunogenicity and protectivity of a new liposomal hepatitis A vaccine

Dileep Padinjarae Vangasseri a, Leaf H. Immunostimulation mechanism of LPD nanoparticle as a vaccine carrier

Coating of mannan on LPD particles containing HPV E7 peptide significantly enhances immunity against HPV-positive tumor

Nanoparticulate STING agonists are potent lymph node-targeted vaccine adjuvants

Adjuvant activity of immunopotentiating reconstituted influenza virosomes (IRIVs)

Virosome-mediated delivery of protein antigens to dendritic cells

Pharmaceutical aspects of intranasal delivery of vaccines using particulate systems

Influenza virosomes as vaccine adjuvant and carrier system

Influenza virosomes supplemented with GPI-0100 adjuvant: a potent vaccine formulation for antigen dose sparing

Stabilization of polyion complex nanoparticles composed of poly(amino acid) using hydrophobic interactions

Immunogenicity of novel nanoparticle-coated MSP-1 C-terminus malaria DNA vaccine using different routes of administration

Nanoparticle vaccines

Liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response

Induction of potent adaptive immunity by the novel polyion complex nanoparticles

Biodegradable nanoparticles composed of dendrigraft poly-L-lysine for gene delivery

An investigation of the factors controlling the adsorption of protein antigens to anionic PLG microparticles

Handbook of biologically active peptides

Polyacrylate dendrimer nanoparticles: a self-adjuvanting vaccine delivery system

Selfadjuvanting polymer-peptide conjugates as therapeutic vaccine candidates against cervical cancer

Lipid core peptide system for gene, drug, and vaccine delivery

Structure-activity relationship of a series of synthetic lipopeptide self-adjuvanting group A streptococcal vaccine candidates

A novel synthetic adjuvant enhances dendritic cell function

Peptidebased subunit vaccine against hookworm infection

Structure/function studies on QS-21, a unique immunological adjuvant from Quillaja saponaria

Structural and immunological characterization of the vaccine adjuvant QS-21

A strong CD8+ T cell response is elicited using the synthetic polypeptide from the C-terminus of the circumsporozoite protein of Plasmodium berghei together with the adjuvant QS-21: quantitative and phenotypic comparison with the vaccine model of irradiated sporozoites

Advances in saponin-based adjuvants

The ISCOM: an immunostimulating complex

ISCOMs and other saponin based adjuvants

Functional aspects of iscoms

Iscom, a delivery system for parenteral and mucosal vaccination

Inulin isoforms differ by repeated additions of one crystal unit cell

The polysaccharide inulin is characterized by an extensive series of periodic isoforms with varying biological actions

Delta inulin: a novel, immunologically active, stable packing structure comprising beta-D-[2->1] poly(fructo-furanosyl) alpha-D-glucose polymers

Inulin crystal initiation via a glucose-fructose cross-link of adjacent polymer chains: atomic force microscopy and static molecular modelling

Advax, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses

Delta inulin polysaccharide adjuvant enhances the ability of split-virion H5N1 vaccine to protect against lethal challenge in ferrets

An inactivated Vero cell-grown Japanese encephalitis vaccine formulated with Advax, a novel inulin-based adjuvant, induces protective neutralizing antibody against homologous and heterologous flaviviruses

JE-ADVAX vaccine protection against Japanese encephalitis virus mediated by memory B Cells in the absence of CD8+ T cells and pre-exposure neutralizing antibody

An inactivated cell culture Japanese encephalitis vaccine (JE-ADVAX) formulated with delta inulin adjuvant provides robust heterologous protection against West Nile encephalitis via cross-protective memory B cells and neutralizing antibody

A novel hepatitis B vaccine containing Advax, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing

Induction of mucosal and systemic antibody and T-cell responses following primeboost immunization with novel adjuvanted human immunodeficiency virus-1-vaccine formulations

Advax-adjuvanted recombinant protective antigen provides protection against inhalational anthrax that is further enhanced by addition of murabutide adjuvant

Severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology

Novel nanoparticle vaccines for listeriosis

A gold glyco-nanoparticle carrying a listeriolysin 0 peptide and formulated with Advax delta inulin adjuvant induces robust T-cell protection against listeria infection

Improving the dromedary antibody response: the hunt for the ideal camel adjuvant

Randomized clinical trial of immunogenicity and safety of arecombinantHlNl/2009 pandemic influenza vaccine containing Advax polysaccharide adjuvant

Immunogenicity and safety of Advax, a novel polysaccharide adjuvant based on delta inulin, when formulated with hepatitis B surface antigen: a randomized controlled Phase 1 study

Immunotherapy-2076. A controlled study of delta inulin-adjuvanted honey bee venom immunotherapy

Delta inulin adjuvant enhances plasmablast generation, expression of activation-induced cytidine deaminase and B-cell affinity maturation in human subjects receiving seasonal influenza vaccine

A single immunization with inactivated H1N1 influenza vaccine formulated with delta inulin adjuvant (Advax) overcomes pregnancy-associated immune suppression and enhances passive neonatal protection

Advax delta inulin adjuvant overcomes immune immaturity in neonatal mice thereby allowing single-dose influenza vaccine protection

Safety and immunogenicity of a delta inulin-adjuvanted inactivated Japanese encephalitis virus vaccine in pregnant mares and foals

Enhanced pulmonary immunization with aerosolized inactivated influenza vaccine containing delta inulin adjuvant

Mannose receptor-mediated gene transfer into macrophages using novel mannosylated cationic liposomes

HIV-1-specific cell-mediated immune responses induced by DNA vaccination were enhanced by mannancoated liposomes and inhibited by anti-interferon-gamma antibody

Development of an antigen-presenting cell-targeted DNA vaccine against melanoma by mannosylated liposomes

Mannosylated niosomes as adjuvant-carrier system for oral genetic immunization against hepatitis B

Mannosylated niosomes as carrier adjuvant system for topical immunization

Chitosan-based systems for the delivery of vaccine antigens

Chitosan-based nanoparticles for improving immunization against hepatitis B infection

The concomitant use of the LTK63 mucosal adjuvant and of chitosan-based delivery system enhances the immunogenicity and efficacy of intranasally administered vaccines

Encapsulation of adenoviral vectors into chitosan-bile salt microparticles for mucosal vaccination

Phase I trial of ImmTher, a new liposome-incorporated lipophilic disaccharide tripeptide

Immunologic and toxicologic study of disaccharide tripeptide glycerol dipalmitoyl: a new lipophilic immunomodulator

Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles

Structurebased programming of lymph-node targeting in molecular vaccines

Development of soluble inulin microparticles as a potent and safe vaccine adjuvant and delivery system

Biodegradable nanoparticle delivery of a Th2-biased peptide for induction of Thl immune responses

Molecularly engineered poly(ortho ester) microspheres for enhanced delivery of DNA vaccines

Oxidation-sensitive polymeric nanoparticles

Incorporation controlled release of silyl ether prodrugs from PRINT nanoparticles