Nanoparticles for Immune Stimulation Against Infection, Cancer, and

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Nanoparticles for Immune Stimulation Against Infection, Cancer and Autoimmunity Santiswarup Singha, Kun Shao, Kristofor Kenneth Ellestad, Yang Yang, and Pere Santamaria ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05950 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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Nanoparticles for Immune Stimulation Against Infection, Cancer and Autoimmunity

Santiswarup Singha1*, Kun Shao1*, Kristofor K. Ellestad1, Yang Yang1,2 and Pere Santamaria1,3

1Julia

McFarlane Diabetes Research Centre (JMDRC) and Department of Microbiology,

Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada 2Department

of Biochemistry and Molecular Biology, Cumming School of Medicine, University of

Calgary, Calgary, Alberta, T2N 4N1 Canada 3Institut

D’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, 08036, Spain

*Contributed equally to this work

Contact information: P. Santamaria ([email protected]) or Y. Yang ([email protected])

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ABSTRACT Vaccination using nanocarrier-based delivery systems has recently emerged as a promising approach for meeting the continued challenge posed by infectious diseases and cancer. A diverse portfolio of nanocarriers of various sizes, compositions, and physical parameters have now been developed, and this diversity provides an opportunity for the rational design of vaccines that can mediate targeted delivery of various antigens and adjuvants or immune regulatory agents in ways unachievable with classical vaccination approaches. This flexibility allows control over the characteristics of vaccine-elicited immune responses such that they can be tailored to be effective in circumstances where classical vaccines have failed. Furthermore, the utility of nanocarrier-based immune modulation extends to the treatment of autoimmune disease where precisely targeted inhibition of immune responses is desirable. Clearly, the selection of appropriate nanocarriers, antigens, adjuvants, and other components underpins the efficacy of these nano-immune interventions. Herein we provide an overview of currently available nanocarriers of various types and their physical and pharmacological properties with the goal of providing a resource for researchers exploring nanomaterial-based approaches for immune modulation and identify some information gaps and unexplored questions to help guide future investigation.

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KEYWORDS: nanoparticles, nano-design, nanovaccine, infectious diseases, cancer, vaccine, immune modulation, translational potential VOCABULARY: Pattern recognition receptor (PRR): Receptor for a conserved molecular pattern associated with pathogens or tissue destruction. Pathogen-associated molecular pattern (PAMP): A conserved molecular pattern present on microbes that the immune system has evolved to recognize as a hallmark of pathogen presence. Damage-associated molecular pattern (DAMP): Host endogenous molecules that are expressed or released upon tissue damage to induce the immune response. Toll-like receptor (TLR): A family of transmembrane PRR that play an important role in sensing pathogens as well as endogenous danger signals and triggering inflammation. Stimulator of Interferon gene (STING): This is a signaling molecule that senses cytosolic DNA and controls the transcription of several host defense genes such as type I interferons. Nanovaccines: Nanoformulations that are used for induction of antigen-specific immune responses.

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Vaccination represents one of the most successful public health interventions ever introduced. The power of vaccines comes from their ability to mobilize the immune system and induce long-term immunological memory. Upon pathogen exposure in an immunized host, primed antigen-specific immune cell populations, including effector CD4+ and CD8+ T cells and antibody-producing B cells, expand rapidly to eliminate pathogens before their extensive propagation and dissemination in the host, avoiding the pathology associated with infection. Despite their successes, the development of vaccines against emerging infectious diseases, such as HIV and pandemic influenza, has proven to be challenging because these pathogens have evolved mechanisms of evading or misdirecting the immune response.1 Furthermore, as tumor cells are self-derived and adept at mediating immune suppression, reliable therapeutic induction of effective tumor-specific primary immune responses remains elusive. Breaking immune suppression non-specifically (e.g. with anti-PD-1, anti-CTLA-4 mAb therapy) potentiates existing anti-tumor immune responses and induces tumor regression in some patients, but often leads to adverse auto-inflammatory events.2 In the case of human autoimmunity, there is a need to develop interventions that bolster organ/tissue specific immune regulation and tolerance without compromising systemic immunity. Traditional vaccines mostly consist of attenuated pathogens as a source of both target antigens and adjuvants. However, for vaccines using subunits or peptides of pathogen-associated antigens to alleviate the safety concerns associated with attenuated/killed pathogens, incorporation of adjuvants into the formulation becomes essential for effectiveness.3 Adjuvants are mostly ligands of families of pattern-recognition receptors (PRR) that recognize evolutionarily conserved molecular patterns of microbes (pathogen associated molecular patterns, PAMPs) or tissue damage (damage associated molecular patterns, DAMPs). These receptors are expressed on the surface or in subcellular compartments of innate immune cells, including neutrophils, macrophages, natural killer (NK) cells and dendritic cells (DCs). Adjuvant/ligand engagement of PRRs triggers activation and “maturation” of innate immune cells, resulting in the release of various cytokines and chemokines

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and up-regulation of molecules associated with antigen presentation. Mature DCs migrate to lymph nodes where they present antigens and co-stimulatory signals to prime naïve T cells. Ideally, immune responses should be qualitatively and quantitatively fine-tuned to deal appropriately with specific host insults. In this setting, adjuvants can have a profound effect on the types of immune responses that arise in response to vaccination. PRRs consist of multiple families of non-phagocytic receptors, including Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which recognize a wide variety of extracellular or endosomal PAMPs and DAMPs, as well as retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) and cytosolic DNA sensors such as stimulator of IFN genes (STING).4,5 The engagement of individual ligands with PRRs triggers cascades of signaling events that can potentiate different types of antigen-specific regulatory or effector responses. Ligation of TLR2 preferentially facilitates Th2 responses, while signals from TLR4 favour the development of Th1 responses.6 As the most widely used adjuvant for more than a half century, alum primarily induces Th2 responses and antibody production without effectively promoting cytotoxic T-lymphocyte (CTL) responses.3 In contrast, LPS (a ligand of TLR4), class A CpG oligodeoxynucleotides (ODN, a ligand of TLR9) and cyclic dinucleotides (CDNs, ligands of STING) stimulate IFN production and elicit Th1 and CTL responses when used as adjuvants. In addition, targeted delivery of identical adjuvants to subsets of DCs could lead to a Th1 or Th2 response bias.7 Furthermore, TLR signals have also been reported to impact regulatory T cell responses.8,9 It therefore stands to reason that approaches allowing accurate delivery of sufficient doses of antigen and selected adjuvants and/or other immune-modulating agents to specific subsets of innate and/or adaptive immune cells will emerge as leading therapeutic interventions not only for infectious diseases but also cancer10 and autoimmune diseases. In the context of infection and cancer, the next generation of vaccines/interventions shall be capable of suppressing antigenspecific immunoregulation in addition to promoting effector responses. Whereas such challenges

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have not been adequately addressed with classical vaccine designs, nanotechnology-based vaccines are emerging as preferential options. In the past decade, nanovaccines comprised of a diverse range of core/carrier structures and materials have been developed and tested for efficacy. These nanocarriers possess very different chemical and physical properties, some of which, including size, surface chemistry and internal “payload”, can be fine-tuned for specific purposes. The immune stimulating properties - in particular the adjuvanticity of these nanocarriers - have also been intensively investigated. In addition, approaches that enable the conjugation and/or loading of various antigens, adjuvants, and other immunomodulatory reagents to the surface of or within encapsulated spaces of nanocarriers have been developed and explored.

Rapid developments in this multidisciplinary field, lying at the

interface between nanotechnology, materials science and immunology will enable the development of nanovaccines with optimized pharmacokinetic properties, minimal off-target effects and maximal potency. This review outlines the current state-of-the-art in this area, with a focus on nanocarrier types, antigen/adjuvant loading approaches, and principles for designing effective nanotechnologybased immune interventions.

Nanocarriers Ideally, an optimal carrier should be able to protect vaccine materials from degradation, to promote the uptake of those materials by the appropriate professional antigen-presenting cell (APC) types, and to enable the release of the payload in a manner that promotes the orchestration of an effective and specific immune response against the desired target(s). Different types of nanostructures, organic, inorganic or biological material-based, have been developed for this purpose (Table 1 and Figure 1). Organic nanoparticles (NPs). A variety of polymeric organic NPs have been developed as vaccine carriers mostly because of their biocompatibility and manufacturing simplicity. Many of these

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materials allow self-assembly in the presence of antigens and adjuvants. The size of the polymeric NP designs that have been used vary widely, from very small (20-30 nm) to large (>500 nm). As antigen carriers, large porous poly(L-lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) microparticles (500-800 nm) loaded with hepatitis B antigens and delivered via the pulmonary mucosal route induced antigen-specific humoral responses. Whereas DCs preferentially phagocytosed particles smaller than 500 nm in diameter, larger particles were primarily uptaken by macrophages.11 Antigenic peptides and proteins, as well as adjuvants, can be encapsulated into PLGA particles, allowing slow release of antigen at the target site, prolonged antigen presentation by DCs ( Figure 2) and a sustained immune response.12 Thiol-(-SH) modified recombinant malarial antigen was conjugated to the surface of maleimide-functionalized PLGA particles and, when given in the presence of an adjuvant, these NPs elicited high titer antibody responses with increased antibody affinity as compared to a mixture of non-NP-conjugated antigen and adjuvant.13 When anthrax antigen was encapsulated into PLGA particles (~250 nm in diameter), vaccination elicited Th1/Th2 CD4+ T-cell responses and the production of antigen-specific IgG1 and IgG2a antibodies, leading to improved animal survival as compared to non-NP-based immunization14 When CpG oligonucleotide (as adjuvant) was co-encapsulated with tetanus toxin in PLGA particles, vaccination induced both Th1 cellular responses and greatly intensified antibody responses.15 PLGA particles are FDA-approved vaccine carriers for clinical application. However, PLGA particles are unstable and their size is difficult to control. The importance of NP size is highlighted by the finding that small pluronic-stabilized polypropylene sulfide (PPS) NPs ( 8 mg/kg) and GNPs also upregulate production of cytokines, such as IL-1, IL-6, and TNF by macrophages.30 The risks and long term consequences of the use of GNPs as vaccine carriers requires further investigation. Iron oxide nanoparticles (IONP) have been extensively used for imaging purposes, drug delivery, and more recently vaccination. Stable IONPs can be synthesized through various chemical processes and a number of IONP products have been approved for clinical application.31 A classical synthesis method is alkaline co-precipitation. This approach can produce IONPs at high yields but affords limited control over particle size. Thermal decomposition generates IONPs of homogeneous size, but the process usually relies on the use of organic surfactants that may pose challenges for functionalization or biocompatibility.32 Surface functionalization underpins payload capacity and the pharmacokinetic properties of IONP in the circulation. Successful functionalization also improves stability and biosafety, as well as the immunological properties of IONPs. Ferumoxytol is a carbohydrate-coated IONP type approved for the treatment of iron deficient anemia and as an MRI contrast agent which, interestingly, has been shown to inhibit tumor growth by inducing M1 polarization of macrophages in animal models.33 A commercially available carboxyl-functionalized 10


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IONP was likewise shown to activate macrophages.34 Immunization of conjugates of this IONP and a malarial antigen (rMSP1) generated strong antigen-specific antibody responses in the absence of other adjuvants.34 Mixing lipo-PEG and lipopeptides with hydrophobic IONPs generated hydrophilic IONPs displaying 23 antigenic peptides on the surface of individual NPs. Because of their small size (~32 nm), these NPs efficiently drained into LNs, were captured by LN DCs and elicited high-titer antibody responses.35 Whereas IONPs co-delivering antigen with the TLR9 ligand CpG induced CD8+ T-cell reponses, delivery of antigen alone exclusively induced antibody responses.36 We have recently developed a small IONP type (20 nm) via a simple, surfactant-free, onestep thermal decomposition reaction that yields water-soluble NPs. This type of IONP (PF-NP) is highly stable in aqueous buffers at physiological pH and osmolarity and can be conjugated covalently with protein, peptide and other ligands via various chemistries. The payload capacity of PF-NP approaches 1 mg of 50 kDa protein/mg Fe (about 70 molecules/NP) or >700 15-20 amino acid-long peptides/NP37 (and unpublished data). High payload capacity is a key attribute for some immunological applications, such as for triggering the formation and expansion of antigen-specific regulatory T-cells using peptide-major histocompatibility complex (pMHC)-based nanomedicines to treat autoimmune inflammation, a process that is critically dependent on pMHC ligand density.37 Another important property of PF-NPs is that they can be readily captured by DCs and macrophages without triggering cellular activation, without accumulating in tissues and organs and without causing detectable toxicity as determined using multiple in vitro and in vivo readouts.37 These properties make PF-NPs an excellent platform with which to build immunoregulatory and immunostimulatory nanomedicines. Many inorganic nanoformulations have intrinsic adjuvant activities. Alum is a classic vaccine adjuvant that enhances antibody responses with limited CTL-triggering effects.38 However, alum NPs (alpha-alumina) markedly enhanced CTL responses via an autophagy pathway in DCs.39 The adjuvanticity of calcium phosphate NPs has been demonstrated in various vaccine formulations and clinical trials have validated their safety.40 Apart from delivering polypeptide antigens, calcium 11


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phosphate NPs have also been evaluated both as adjuvants and as vehicles for DNA vaccines.41 Though they can be prepared using a simple co-precipitation method, these calcium phosphate particles are size heterogeneous (50-400 nm diameter range). Silica nanoparticles (SNP) were initially developed as drug delivery vehicles and their synthesis, functionalization, biocompatibility, toxicity, bio-distribution and controlled release properties have been extensively investigated. Recent studies have demonstrated that SNP, which can be synthesized in a wide range of shapes and sizes with variable porosity and payload capacity, can be effective carriers of antigen and adjuvant to drive both humoral and cellular immune responses.42,43 Whereas surface coating with various molecules allows enhanced cellular targeting and uptake, their porous spaces can be loaded with active biomolecules. SNPs of small size (30 nm) were developed for lymph node-targeted antigen/adjuvant delivery.44 SNPs have also been formulated for oral vaccination.45 SNPs have been used as templates for the production of carbon NPs (CNPs), which also possess a mesoporous structure and can be loaded with large quantities of antigen. These NPs, which appear to have intrinsic adjuvant effects, have been used for oral vaccination, enhancing IgG1 and IgG2a as well as IgA responses.46 In addition to CNPs, various forms of carbon nanotubes (CNT), including single- (SWNT) and multiple-walled (MWNT) nanotubes were tested as platforms for vaccination. The dimensions of these nanotubes range from 50 nm to >1000 nm in length and 2 nm to 50 nm in diameter. CNTs have adjuvant properties and trigger the activation of macrophages and DCs and the production of various cytokines, such as IL-1β, IL-6, and TNF.47 However, whether the irregular shape and/or the strong pro-inflammatory effects of CNTs on immune cells can cause potential toxicity in vivo largely remains to be investigated. A recent report suggests that CNTs administered at doses > 5 mg/kg led to significant mortality in mice, suggesting an abundance of caution.48 Zinc oxide nanoparticles (ZNP), extensively used in the food and cosmetic industries, have immune-stimulatory effects in mice. Antigens can be conjugated to ZNP via zinc binding peptide 12


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(ZBP).49 Though peptide-coated ZNPs became easily aggregated, immunization with antigencoupled ZNPs generated protection against Orientia tsutsugamushi infection by eliciting the formation of antigen-specific antibodies and IFN-secreting CD4+/CD8+ T-cells.49 Biological NPs. As natural biopolymers, various protein molecules such as albumin, gelatin, ferritin, and zein have been used to synthesize NPs for drug delivery. Protein NPs show excellent solubility in aqueous solutions, biocompatibility, and biosafety often with relatively low adjuvanticity. Protein NPs can be made using simple de-solvation and crosslinking, and protein molecules can be modified for conjugation of antigens or targeting reagents.50 However, this method affords limited control over NP size. In contrast, NPs formed by recombinant proteins that can structurally selfassemble offer the benefit of size homogeneity. In addition, their subunits can be genetically fused with antigens. A recombinant ferritin from H. pylori fused with influenza hemagglutinin selfassembled into 20 nm NPs composed of 24 subunits. Vaccination with these HA-NPs generated much stronger humoral anti-influenza responses than inactivated influenza virus, and HA-NP vaccination protected animals from even highly divergent viral strains.51,52 Recently, double-layered protein NPs consisting of a core of recombinant influenza matrix protein M2e displaying a shell of “headless” HA have been further developed and these protein NPs confer potent long-term protective immunity.53

Because both the self-assembled core and shell are conserved viral

components, this new type of nanovaccine minimizes the risk of off-target immune responses caused by ferritin or other non-viral components. Indeed, these protein NPs may represent candidates to replace current seasonal flu vaccines. Self-assembling protein NPs containing five CD8+ HLA-A0311 supertype-restricted epitopes of Toxoplasma gondii antigen, Pan-DR T helper epitopes (PADRE) for CD4+ T-cells, and flagellin as both a scaffold and TLR5 agonist, facilitated presentation of the corresponding peptides to CD4+ and CD8+ T-cells in mice.54 Targeted delivery of biological NPs has also been documented: caveolin protein forms “caveospheres” of 45-50 nm which can be coated with specific antibody molecules for cell-specific delivery.55

The suitability of protein NP

morphologies other than spheres have also been explored. β-sheet fibrillizing peptide 13


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QQKFQFQFEQQ (Q11), chemically modified to allow selective conjugation to properly folded fusion proteins, self-assembled into unbranched nanofibers.56,57 Although the self-assembling peptide itself was non-immunogenic, the antigen-fused peptide induced strong antigen-specific antibody responses in the absence of adjuvant, indicating adjuvanticity as an emergent property of the assembled nanofiber. Lipid-linked peptides that self-assembled into cylindrical micelles displaying an epitope of a model tumor antigen also induced strong CD8+ T cell responses and inhibited tumor growth.58 Similar to nanofibers formed with self-assembling peptides, this nanocylindrical structure also appears to possess an adjuvant effect.58

Finally, viral-like-particles (VLP), consisting of

recombinant viral structural proteins, represent another self-assembling nanovaccination platform that can be used to induce both cellular and humoral immune responses against various pathogens.59 VLPs formulated with hemagglutinin (HA) and matrix 1 (M1) (structural proteins of influenza virus) with IL-12 as adjuvant, generated excellent protection against influenza A/Hong Kong/68 (H3N2) virus.60 The efficiency of VLPs can be ‘tuned’ by employing different adjuvants such as 5′-triphosphate-containing RNA (5′pppRNA), an agonist of the cytosolic viral RNA sensor RIG-I (retinoic acid-inducible gene I).61 VLPs are generally packed and assembled in cellular protein expression systems. Since they cannot propagate, they are much safer than traditional attenuated virus vaccines. A number of studies further demonstrated the potential for efficient self-assembly in cell-free systems of stable VLPs that can serve as vaccine carriers.62,63 The development and characterization of diverse nanocarriers, including organic, inorganic and biological NPs continues at rapid pace, presenting a range of options for meeting the functional requirements associated with individual nanovaccines or immune-modulatory therapies. While in the long run such diversity will clearly benefit the clinical translation of nanovaccination, it should be noted that for many nanocarriers the in-depth analytical information required to support entry into the clinic, including their antigen/adjuvant loading capacity, safety profile and pharmacokinetics, remains limited. As mentioned previously, an abundance of caution is particularly warranted for nanocarriers with strong intrinsic adjuvant activity due to potential toxic effects. 14


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Figure 2. Nanoparticle-induced immune responses for effective vaccination. Antigen presenting cells engulf antigen/adjuvant loaded nanoparticles which accumulate in phagosomes or endosomes for further processing. The cytosolic release of antigen (e.g. via endosomal escape) promotes presentation of antigen to cytotoxic T-cells (CD8+ T-cells, CTL) via the MHC class-I pathway. Endosomal processing of antigen largely initiates the loading of antigen on MHC class-II for subsequent presentation and recognition by T-helper cells (CD4+ T-cells). Cytosolic antigen is processed through proteasomal degradation resulting in precursors which are then transported to the endoplasmic reticulum (ER) resulting in peptides of 8-10 amino acids for loading onto MHC-Class I. MHC Class-II is complexed with an invariant chain and undergoes proteolytic cleavage to produce Class II-associated invariant chain peptide (CLIP) which is replaced by high affinity peptides (~1016 amino acids long), produced from the lysosomal processing of nanoparticle-borne antigen.

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Appropriate control over cytosolic release of antigen/adjuvants is therefore an important consideration for nanovaccine design to facilitate induction of CTL responses.

Incorporating adjuvants into nanocarriers Adjuvants are a key element underpinning vaccine efficacy, yet most adjuvants approved for clinical application are effective at magnifying humoral but not cellular immune responses.3 For vaccines against chronic infections as well as tumors, success will require the evocation of strong cellular immune responses. Many adjuvants under development show promise in this regard, however, the application of these adjuvants is often restricted by instability, off-target activity, and systemic cytokinemia when used at relatively high doses.64-67 Incorporation of adjuvants into nanocarriers (Table 2) can improve adjuvant stability and facilitate targeted delivery (Figure 3) to increase potency and minimize unwanted side effects. Thus, rational design of successful nanocarrier-based vaccines will involve both the selection of optimal adjuvants to guide the orchestration of desired immune responses and the development of methods to incorporate various adjuvants into these nanostructures. The ligation of TLRs by PAMPs results in the activation of innate immunity, eliciting signals that trigger and shape adaptive immune responses68 (Figure 3). Various approaches have been exploited to incorporate TLR agonists into different nanocarriers, including double stranded RNA for TLR3, viral or bacterial single-stranded RNA or Imiquimod, a heterocyclic small molecule agonist for TLR7, unmethylated DNA oligonucleotides containing CpG motifs for TLR9, or lipopolysaccharide (LPS) derivatives for TLR4. Because of the negative charge of nucleoside analogues (dsRNA, ssRNA, DNA), a simple approach is to complex anionic agonists with cationic polymers or inorganic materials via electrostatic interactions. For instance, negatively charged poly (I:C) and a model antigen (OVA) were loaded to positively charged poly (g-glutamic acid, g-PGA) NPs modified with amine moieties. NP loading facilitated uptake of poly(I:C) and OVA antigen by DCs and macrophages and enhanced 16


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retention of antigen and adjuvant in lymph nodes to boost antigen-specific responses.69 CpG-ODN (cytosine-phosphate-guanine-oligodeoxynucleotide), a TLR9 agonist, was physically adsorbed onto the surface of PEG-PE and PSA hybrid micelles, lipid nanocapsules, and gelatin NPs.27, 70,71 The simple surface attachment of adjuvants on nanocarriers effectively abolished systemic inflammatory responses associated with injection of free adjuvants but enhanced immune responses specific to the antigens co-loaded inside or coated on the surface of the nanocarriers. CpG-ODN has also been chemically modified for conjugation with nanocarriers. Sulfhydryltagged CpG-ODNs were conjugated to GNPs via the Au-S bond.72 Aldehyde-terminated CpG was covalently conjugated to the internal surface of non-viral pyruvate dehydrogenase E2 protein NPs using N-β-maleimidopropionic acid hydrazide (BMPH) as a linker.24, 73 Cholesterol-modified CpG was inserted into the lipid components of high-density lipoprotein (sHDL) nanodiscs (10.5 nm). Such nanodiscs enhanced CpG accumulation in lymph nodes and elicited robust anti-tumoral T-cell responses that were superior to those induced by soluble vaccines, inhibiting tumor growth.74 Hydrophobic interactions can also be leveraged for adjuvant loading into/onto nanocarriers. Lipid-like adjuvants, such as the TLR4 agonists monophosphoryl lipid A (MPLA) and LPS, can be associated with lipid components of NPs. Vaccination with MPLA-liposome NPs bearing encapsulated fibroblast growth factor (bFGF) efficiently stimulated bFGF-specific antibody production as compared to bFGF/Complete Freund's adjuvant-vaccinated animals, leading to inhibition of angiogenesis-dependent lung tumor metastasis.75

More recently, Verbeke et al.

embedded MPLA into the lipid bilayer of a liposome-based mRNA vaccine and achieved high levels of T-cell immunity.76 MPLA was also incorporated into a lipid-coating on zinc phosphate hybrid NPs that carried tumor-associated antigenic peptides. Both prophylactic and therapeutic vaccination elicited enhanced CD8+ T-cell responses and anti-tumor effects.77 The TLR7 agonist Imiquimod (R837) is a heterocyclic small molecule with water solubility < 2 mg/mL.

By virtue of its

hydrophobicity, R837 could be engineered into the cores of micelles, the liposomal bilayer, or PLGA copolymers.78,79 In addition, a detoxified LPS derivative was encapsulated in PLGA-based NPs 17


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together with the chemotherapeutic agent paclitaxel. Treatment with such PLGA NPs resulted in significantly increased tumor infiltration by immune cells, including macrophages, DCs and both CD4+ and CD8+ T-cells.80 Coupling adjuvants and antigen to the same nanocarrier is an effective vaccine strategy, delivering antigen and adjuvant simultaneously to the same APCs to promote an optimal T-cell response.74,75,

77, 81,82

The co-coupling of antigen and adjuvant on microspheres significantly

enhances the resulting CTL responses as compared to separate coupling.83 However, co-coupling of antigen and adjuvant is not always crucial. Mohsen et al. packaged adjuvant (CpGs) and antigen into separate VLPs (30 nm) and administration of the VLP mixture generated CTL responses similar to those obtained using VLPs carrying co-packaged adjuvants and antigens. In vivo trafficking experiments demonstrated that both VLPs migrated to the same lymph nodes.84 Cyclic dinucleotides (CDNs) and their synthetic derivatives, including cyclic di-GMP (cdGMP), 2’, 3’-cyclic-GMP-AMP (2',3'-cGAMP), cyclic di-inosine monophosphate, and cyclic diAMP (cdAMP), act as strong adjuvants by binding to the cytoplasmic DNA sensor STING and triggering type I interferon (IFN) production.85

However, the pharmacokinetic properties and

systemic distribution of these small molecule STING agonists as vaccine adjuvants raises a safety concern – they tend to accumulate in the bloodstream rather than lymph nodes, promoting systemic inflammation.66 Nanocarrier formulations containing STING agonists have exhibited potent APC activation properties, eliciting robust and durable cellular and humoral responses.66, 86 Delivery of CDNs (cdGMP and cdAMP) via liposomes promoted adjuvant transport through lymphatics into draining lymph nodes.87

In addition, cationic nanomaterials such as poly (beta-amino esters)

(PBAEs) form nano-complexes with CDNs through electrostatic adsorption.88 Liposomes loaded with cdGMP drove a 5-fold increase in vaccine-specific CD4+ T-cell expansion as compared to unformulated CDNs and enhanced anti-tumor immunity.66 It has also been shown that, when used as antigen carriers, polymers containing tertiary amines with cyclic side chains, such as PC7A, bind directly to STING and trigger strong CTL and anti-tumor responses without triggering systemic 18


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cytokinemia.89 In addition, PEG-b-PC7A self-assembled into ultra-pH-sensitive micelles that could be efficiently loaded with cGAMP via hydrophobic interactions and an internal salt-bridge between PEG-b-PC7A and cGAMP.90 These pH sensitive liposomes were designed to achieve effective cytosolic delivery of STING agonists, releasing cGAMP upon endo-lysosomal acidification with subsequent successful endosomal escape.86,

90,91

Even STING agonists encapsulated in acid-

sensitive acetylated dextran (Ace-DEX) polymeric microparticles enhanced type-I IFN responses up to 50-fold in vivo, demonstrating that effective cytosolic delivery of STING agonists can have very powerful effects on vaccination outcome.86 Clearly, STING agonists are promising adjuvants to elicit both cellular and humoral responses against poorly immunogenic targets. However, they remain a relatively new class of adjuvants, and the range of nanoformulations that have been employed to deliver these adjuvants is rather limited – for example, no inorganic nanocarrier formulations for STING agonist delivery have yet been reported. Further development of NP-based approaches to deliver STING and other innate PRR agonists will undoubtedly lead to enhanced nanovaccine efficacy.

Incorporation of non-adjuvant immune modulators into nanocarriers Nanocarriers can also be used as vehicles to co-deliver antigen with immuno-modulatory molecules, as a way to promote antigen-specific immunoregulation. A natural agonist of aryl hydrocarbon receptor (AhR), ITE (2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester), was incorporated into the PEG layer of GNPs with specific autoimmune disease-relevant antigenic peptides.92 As a transcription factor, AhR is expressed in DCs and T-cells, and translocates into the nucleus upon ligand-induced activation, triggering expression of a specific set of genes. Uptake of ITE/antigen-bearing GNPs by DCs triggered the acquisition of a tolerogenic phenotype, promoting FoxP3+ regulatory T-cell formation. Antigen-specific T- and B-cell tolerance was also induced in animal models of experimental autoimmune encephalomyelitis (EAE), allergy, and anti-factor VIII-associated hemophilia by 19


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treatment with PLGA particles encapsulated with the immune modulator rapamycin and the corresponding autoantigens.93 Tolerance was only induced by particle-encapsulated, but not free rapamycin. Particles encapsulated with antigen or rapamycin alone were similarly ineffective for tolerance induction.

In vivo, NPs were captured by DCs, macrophages and B cells, strongly

suggesting a role for APCs in tolerance induction.

Figure 3. Differential pattern recognition receptor engagement by nanoparticle-borne adjuvants depending on subcellular localization. Cells, and particularly antigen presenting cells, express a variety of PRR localized to various cellular compartments, including the plasma membrane, endosomes, and cytoplasm. Depending on the compartment to which NP gain access, differential responses to NP and/or NP-borne adjuvants can vary depending on downstream signaling from co-localized PRR, potentially resulting in markedly different outcomes in terms of the magnitude and phenotype of vaccine-induced immune responses.

Direct nanoparticle-based T-cell activation/modulation 20


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The approaches discussed above seek to bolster the efficiency of the types of immune responses elicited by classical vaccination, where induction or modulation of immunity is achieved via activation/modulation of innate immune cell types. This strategy is effective but suffers from certain limitations due to indirect addressal of the adaptive immune cells directly responsible for pathogen or tumor clearance (e.g. T- and B-cells).94 The use of artificial APCs (aAPC), particularly nanoscale aAPCs, is an appealing approach to directly activate/modulate CD8+ and CD4+ T cells with selected antigen specificities, thus overcoming the limitations imposed by the cellular APC “middle man” requirement in conventional vaccination strategies. The design, feasibility, and function of aAPCs have been extensively investigated and evaluated in recent years. aAPCs have been engineered using various carrier platforms, including iron oxide, PLGA polymers, and carbon nanotubes of different shapes and sizes.95-100 Because signals through the Tcell receptor (TCR) for antigen and co-stimulatory molecule CD28 are the minimum requirements for T-cell activation, most aAPC designs involve surface-anchored agonistic anti-CD28 antibodies (to deliver co-stimulatory signals) and peptide-MHC (pMHC) class I molecules or anti-CD3 antibodies (to trigger antigen-specific vs. non-antigen-specific TCR signaling, respectively).95-99 While in most studies pMHC molecules and anti-CD28 antibody molecules were directly conjugated to aAPCs, it was reported that aAPCs generated using an indirect conjugation approach (biotinylated pMHCs/anti-CD28 bound to aAPC via surface-bound anti-biotin or anti-MHC) stimulated cognate Tcells more efficiently, likely because pMHCs on the surface of aAPCs were directionally oriented and had superior spatial flexibility.95 It has been shown that carrier geometry impacts the function of aAPCs.98 Ellipsoid aAPCs activated CD8+ T-cells more efficiently than spherical aAPCs; in fact, CD8+ T-cells preferentially migrated to and interacted with long ellipsoid aAPCs.98 This physical effect is likely associated with aAPC surface area. When nano-sized aAPCs (from 50 to a few hundred nm) and cell-sized microaAPCs were coated with pMHCs, large nano-aAPCs or cell-sized micro-APCs activated T-cells more efficiently than 50 nm aAPCs.97, 100 Whereas large aAPCs (300 nm, 600 nm, and 4.5 m) formed 21


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clusters on the surface of T-cells, 50 nm nano-aAPCs did not. Application of a magnetic field triggered nano-aAPC cluster formation, resulting in strong T-cell activation, suggesting that activation and cluster formation are somehow mechanistically linked.98,

100

Although the authors

concluded that nano-sized aAPCs cannot spontaneously trigger this process, the pMHC densities that were coated onto these compounds were very low. In addition, direct coating of pMHC and antiCD28 antibody on these aAPCs likely hindered spatial flexibility of the molecules, hence the efficiency of antigen receptor-mediated pMHC engagement. We have recently shown that small pMHC-coated NPs (PFM-NPs) can efficiently re-program antigen-specific autoreactive T-cells into autoimmune disease-suppressing regulatory T-cells, by directly ligating antigen receptors on cognate T-cells and promoting cluster formation.37, 101 These NPs have a 20 nm iron oxide core beneath a maleimide-functionalized PEG layer, and an overall hydrodynamic diameter of 40-50 nm. This NP design allows high density-conjugation of pMHCs or other bio-materials onto the NP’s surface, directional exposure of the peptide-binding domain of the coated pMHCs towards T-cells, and considerable spatial flexibility afforded by the 10 nm thick, pMHC-anchoring PEG layer. pMHC-PFM-NPs specifically bind to cognate T-cells triggering the sustained assembly of large NP clusters on their surface. This overcomes the low affinity of the monomeric pMHC-antigen receptor interaction and promotes a prolonged pMHC-NP/T-cell engagement, lasting hours rather than seconds, that results in cooperative signaling among the cytoplasmic tails of neighbouring antigen receptors.37 By experimenting with different NP sizes and pMHC densities, we demonstrated that the ability of pMHC-NP compounds to trigger antigenreceptor and NP cluster formation is strictly a function of pMHC density, such that only NPs coated at densities greater than a specific threshold could trigger such responses.37 Although these NPs were designed to enhance immune regulation as a therapeutic intervention for autoimmune diseases, these findings have implications for the optimal design of aAPCs to elicit immunity, as opposed to immunoregulation.

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Various cytokines have been encapsulated into PLGA-based aAPCs in an attempt to facilitate the activation/expansion of targeted T-cells and guide their differentiation. Interleukin-2 (IL2) encapsulated into PLGA aAPCs was more effective than soluble IL-2 in driving T-cell proliferation in vitro 97 and T-cells expanded in the presence of IL-2-loaded aAPCs had improved anti-tumorigenic activity in vivo.99 Interestingly, encapsulated TGF/IL-2 in CD4+ T-cell targeted PLGA particles was reported to facilitate the induction of FoxP3+ Treg cells.102 It should be noted, however, that the effectiveness of aAPCs has mostly been evaluated in in vitro culture systems, with limited in vivo data. Extensive experimentation in vivo will be needed to further harness the theoretical potential of aAPCs for triggering active immunity against infection and/or cancer.

Concluding remarks The concept of nanovaccination emerged more than 20 years ago.

Although

nanovaccination has not yet successfully entered clinical practice, intensive investigation has led to: 1. The development of synthesis methodologies for nanocarrier platforms based on a variety of materials; 2. A basic understanding of the biophysical and biochemical properties, potential cellular toxicity and immunogenicity of these nanoplatforms; 3. The establishment of approaches and conditions for antigen/adjuvant loading/conjugation into or onto the surface of different nanocarriers; 4. Estimation of antigen/adjuvant loading capacity of different nanoplatforms. These studies have also generated valuable information not only on the pharmacokinetics, biodistribution and systemic toxicity of various nano- and micro-particle-based vaccines, but also on the impact of design variables such as size, shape and surface chemistry on their biological efficacy. This work remains incomplete, but the data available to date have provided valuable guidance for future improvements. The literature provides compelling evidence that slow release of antigens and their protection from degradation by nanocarriers results in prolonged antigen presentation, leading to more sustained and potent immune responses capable of supporting the formation of immunological memory. It is also clear that nanocarriers of different sizes reach professional APCs such as DCs 23


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via different routes. Small sized nanocarriers migrate to and accumulate in lymph nodes, where they are efficiently captured by resident DCs. On the other hand, large nanocarriers are inefficient at infiltrating lymph nodes and are perhaps more likely to be captured by phagocytes elsewhere. Clearly, size is an important nanovaccine design attribute, particularly when considering routes of immunization such as subcutaneous, intravenous and mucosal or oral.103 Furthermore, the cationic surface of organic/inorganic nanocarriers can play a role in their interaction with and subsequent activation of DCs. Surface charge, irregular shape, repeating molecular patterns, and certain chemical properties are likely responsible, at least partially, for the intrinsic adjuvanticity of nanocarriers.22, 104 Nevertheless, packaging exogenous adjuvants into nanocarriers to facilitate their release into specific cellular compartments – such as STING agonists into the cytosol – has proven to enhance vaccine potency. The development of nanocarriers as vaccination platforms has also paved the way for their use as immune modulators to treat autoimmune disease.

Accordingly, several nanocarriers

originally designed for use as vaccines have been adapted as therapeutic devices. These devices, including aAPCs, are designed not only to target innate immune cells, but also to directly engage with subsets of antigen-specific T cells. Because of the low frequency of antigen-specific T-cells especially those recognizing tumor antigens or tissue-specific self-antigens - defining the parameters that are critical for efficacy remains a challenging task. The available data strongly indicate that directional conjugation of ligands such as pMHCs, ligand flexibility on the nanocarrier surface and ligand density play critical roles in defining potency. However, when multiple ligands – such as pMHCs and co-stimulatory molecules – are conjugated to the surface of the same nanocarrier, the relative ratios of the various ligands will undoubtedly contribute to therapeutic efficacy or lack thereof, an issue that has not yet been systematically examined. Furthermore, the nature of the surface coat on these compounds will define their phagocyte affinity, hence their pharmacokinetics; pegylated surfaces, for example, appear to delay phagocyte recognition, binding and intake. In addition, it has been suggested that the absorption of plasma proteins onto these pegylated NPs contributes to 24


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rendering them partially invisible to phagocytes (affording a “stealth” effect).105 It remains to be seen how this information can be harnessed to engineer next-generation nanovaccines. Although nanovaccines have not yet matured to the point of clinical application, the knowledge accumulated from evaluation of individual nanovaccine designs suggests potential vaccination strategies depending on the desired immune response. For example, one might be able to promote a humoral response, a cellular immune response or both, whereas classical vaccines have mostly served to induce humoral (antibody) responses. Vaccination with a combination of selected nanocarriers of different sizes/properties carrying either identical or different epitopes/adjuvants and administered simultaneously via different routes may result in the activation of lymph node DCs at different anatomical locations. Furthermore, it might be possible to combine nanovaccination with the use of aAPCs to enhance or prolong specific adaptive immune responses. In conclusion, the availability of diverse nanocarrier platforms provides a range of options for precision customization of nanovaccines and other nanotechnology-based immune interventions. Continued development of nanocarrier platforms will undoubtedly provide a wealth of future opportunities for preventative and acute immune therapies.

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ACKNOWLEDGMENTS We thank the members of our laboratory for their contributions and insights. We apologize to those authors whose work could not be referenced here owing to space limitations. The authors’ work summarized here was funded by the Canadian Institutes of Health Research (CIHR; FRN 136866), Diabetes Canada (NOD_OG-3-15-4963-PS), the Crohn’s and Colitis Foundation of Canada, the Multiple Sclerosis Society of Canada (MSSC; EGID 2641), ISCIII and FEDER, NEURON7-FP-715018, MINECO (SAF2015-68187-R), and Generalitat de Catalunya (SGR and CERCA Programmes; 2017-SGR-338). The JMDRC was supported by the Diabetes Association (Foothills) and currently by Diabetes Canada.

COMPETING INTERESTS P. Santamaria is scientific founder of Parvus Therapeutics Inc. and has a financial interest in the company.

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Table 1 - Classification, composition, synthesis methods and properties of selected nanocarriers reported in the literature. Types of Nanoparticle

Nano-Carrier

Composition/materials

PLA/PLGA

poly (L-lactic acid) or poly (lactic-co-glycolic acid) poly (D, L-lactic-co-glycolic acid)

PLGA PLGA PLGA

ORGANIC

poly (D, L-lactic-co-glycolic acid) poly (D, L-lactic-co-glycolic acid)

PLGA

poly (D, L-lactic-co-glycolic acid)

PPS

Poly (propylene sulfide)

chitosan– pullulan

Polyanion carboxymethyl pullulan Polycation N-trimethyl chitosan chloride Chitosan chloride Chitosan glutamate Chitosan and Pentasodium triphosphate alginate-polyethylenimine

Chitosan Nanogel

Liposome

ICMV (Interbilayer crosslinked multilamellar vesicles)

Gelatin nanoparticles

INORGANIC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Distearoyl phosphatidylcholine, cholesterol and Stearylamine, 1,2-Dioleoyl-snGlycero-3Phosphocholine, 1,2-di-(9Z-octadecenoyl)sn-glycero-3-phospho-(1'racglycerol), (1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N[4-(p-maleimidophenyl) butyramide) Gelatin type A

Synthesis Methods emulsion solvent evaporation water in oil in water (w/o/w) emulsion emulsion solvent evaporation water in oil in water (w/o/w) emulsion water in oil in water (w/o/w) emulsion Inverse emulsion polymerization

Reference

~500-800 nm

Adjuvant Property -

~300 nm

-

12

290 nm Lipid anchored Maleimide 230 nm and zeta potential -18.8 mV

-

13

-

14

290-308 nm

-

15

~30-100 nm Pluronic or Polyhydroxylated/ polymethoxylated 207-603 nm and zeta potential +14 to +33 mV. Polycationic surface

-

16, 17

-

18

311 nm and zeta potential +30.6 mV 80-120 nm and zeta potential +18 to +39 mV and Cationic surface

-

19

-

20

192-216 nm and zeta potential +53 to + 60 mV, and Cationic surface

-

22

Dispersion of lipid thin film in bis-tris propane

192 nm

-

23

Desolvation and crosslinking

272 nm and Zeta potential + 4 mV, Cationic surface 2-50 nm (EM) 20 nm (EM)

-

27

-

28 37

31- 38 nm and PEG/lipid modified surface. 60 nm and 200 nm

-

35

++

39

polyelectrolyte complexation

ionic crosslinking and coacervation Dropwise addition of alginate solution into polyethylenimine solution Dispersion of lipid thin film in polar solvent

Size and Surface

11

GNP PF-NP

Gold Iron oxide

IONP

Iron oxide

Alum nanoparticles Calcium phosphate nanoparticles SNP (Silica Nanoparticle)

Alpha-alumina

Reduction Thermal decomposition Thermal decomposition -

Calcium phosphate

Co-precipitation

100 nm to less than 1.2 m

++

40, 41

Mesoporous Silicate

Chemical synthesis in presence of base and surfactant Acid catalyzed carbon deposition

90 nm (EM) and 130 nm (EM)

+

43, 45

470 nm and hydrophobic surface.

+

46

Carbon nanoparticle

Mesoporous carbon

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BIOLOGICAL

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on mesoporous silica nanoparticle Chemical synthesis in presence of base and surfactant Self-assembly

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ZNP (Zinc oxide nanoparticle)

Zinc oxide

SAPN Caveospheres

Self-Assembling Protein Nanoparticles Caveolin protein

In-vivo synthesis

~45-50 nm (EM)

-

55

Nano-fiber

β-sheet fibrillizing peptide

Self-assembly

+

56, 57

Peptide micelle

Peptide, anchored with dipalmitic acid.

Self-assembly

+

58

VLP (Viruslike-particle)

Protein nanoparticle

in vivo or in vitro packaging

Width 15 nm and length on the order of microns Cylindrical micelles with 8.0 nm width and 50-300 nm in length 15-120 nm

+

59, 62

28


~200-550 nm and Zeta potential -12 to -30 mV.

-

49

30 nm (EM)

-

54

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Table 2 – Characteristics of various approaches to incorporate adjuvants into nanocarriers Adjuvants

Nanomaterials

Nanoformulation

Encapsulation/Conjugation Approaches

Antigen

Size (nm)

Administration Route

Effects

Refs

MPLA or LPS (TLR4)

Lipid

Hybrid NPs

Fabricated into the building materials

TRP2180– 188; hGP10025–

30

i.d.

Enhancing CD8+ T cell response.

77

Liposomes


bFGF

173

i.m.

75

PLGA

SLN

Encapsulation

N/A

236293

peritumoral

Imiquimod (R837) (TLR7)

PLGA

SLN

Encapsulation

Ovalbumin

140170

In vitro activation of DCs

CpG-ODN or CpG (TLR9)

PLGA

Nanospheres

Encapsulation

Tetanus toxoid

290

s.c.

Lipid

Liposomes

Encapsulation

p-Trp2

4045

s.c.

Chitosancoated lipid NPs

Electrostatic adsorption

N/A

5080

i.t.

Nanodiscs


OVA or Adgpk

10.5

s.c.

NPs

Covalent conjugation

N/A

30

i.t. or i. I. or c. I.

Hybrid micelles

Electrostatic adsorption

Trp2

Nanoparticles for Immune Stimulation Against Infection, Cancer, and

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