Design of nanomaterial based systems for novel vaccine development.

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Cite this: DOI: 10.1039/c5bm00507h

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Design of nanomaterial based systems for novel vaccine development Liu Yang,a Wen Li,b Michael Kirberger,c Wenzhen Liao*a and Jiaoyan Ren*a With lower cell toxicity and higher specificity, novel vaccines have been greatly developed and applied to emerging infectious and chronic diseases. However, due to problems associated with low immunogenicity and complicated processing steps, the development of novel vaccines has been limited. With the rapid development of bio-technologies and material sciences, nanomaterials are playing essential roles in novel vaccine design. Incorporation of nanomaterials is expected to improve delivery efficiency, to increase immunogenicity, and to reduce the administration dosage. The purpose of this review is to discuss the employment of nanomaterials, including polymeric nanoparticles, liposomes, virus-like particles, peptide amphiphiles micelles, peptide nanofibers and microneedle arrays, in vaccine design. Com-

Received 5th November 2015, Accepted 10th February 2016

pared to traditional methods, vaccines made from nanomaterials display many appealing benefits,

DOI: 10.1039/c5bm00507h

including precise stimulation of immune responses, effective targeting to certain tissue or cells, and desirable biocompatibility. Current research suggests that nanomaterials may improve our approach to the

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design and delivery of novel vaccines.

1.

Introduction

1.1 Cellular mechanism of immune responses during vaccination a

College of Light Industry and Food Sciences, South China University of Technology, Uangzhou 510640, China. E-mail: [email protected], [email protected], [email protected]; Fax: (+86)20-38897117; Tel: (+86 )20-87112594 b IHRC, inc., 2 Ravinia Dr NE, Atlanta, GA 30346, USA c Department of Natural Sciences, Clayton State University, 2000 Clayton State Boulevard, Morrow, GA 30260, USA

Liu Yang, a postgraduate student in Food Science and Engineering, South China University of Technology (SCUT). She obtained her bachelors’ degree in biotechnology from South China Agricultural University in June, 2011. After graduation, she worked for Center for Disease Control and Prevention, Guangzhou Military Command, as a research assistant from 2011 to 2015, focusing on Liu Yang Dengue Vaccine study. From September, 2015 till now, she worked on the preparation, purification and functional identification of bioactive peptides.

This journal is © The Royal Society of Chemistry 2016

Vaccination, one of the most effective forms of preventative medicine, offers protection by eliciting pathogen-specific immune responses, and establishing long-term immunologic memory.1 The development of vaccines was responsible for the elimination of smallpox in the 1960s and 1970s, a significant decrease of poliomyelitis in the 1950s, effective control of

Wen Li

Wen Li, Proteomics Laboratory Scientist from IHRC, inc. She graduated as Ph.D. of molecular genetics and biochemistry from Georgia State University. Her research mainly focuses on two areas: (1) Multidrug resistance in cancer: identify the structure and function of ABC drug transporter involved in drug efflux; (2) Identify cell surface antigen targets of microorganisms using genomics, proteomics and bioinformatics analyses.

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measles, rubella and many other infectious diseases.2 However, at present, public health is being threatened by a large number of infectious diseases such as HIV, malaria, tuberculosis and hepatitis C, for which we have no vaccines, or the vaccines that have been developed showed limited efficacy.3 Besides, emerging delivery systems of some existing vaccines that can improve antigen intake, enhance immunogenicity, target specific antigen presenting cells (APCs), optimizing immunization routes, and improving biosafety are urgently needed. Due to the lack of fundamental understanding of the in vivo behavior of nanomaterials as delivery systems, main hurdle of the delivery systems, in most cases, lies in how to effectively elicit cell immune response. Therefore, to design novel vaccines and strategies for better vaccine delivery systems are vital to address these threats.2 The rapid development of bio-technologies and immunology research suggests that future vaccines may be developed with the intent to prevent not only infections, but also chronic diseases, autoimmune diseases, anaphylaxis, and cancers. In the near future, the impact of vaccination, as a medical intervention, may extend beyond prophylactic immunization.4 At present, several active immunotherapies for cancers, aimed at inducing host-specific and tumor-specific immune responses, are provided in clinical settings.5 Novel vaccines may have the potential to overcome limitations of traditional vaccines based on improved immunological specificity, reduced toxicity, and shorter development time.6 A deeper understanding of the immune system will improve the design of novel vaccines, based on our increased understanding of cellular and molecular immunology, including the functions of APCs, T and B lymphocytes, cytokines, and their interactions in the course of adaptive immune response. Research has demonstrated that T and B cell are responsible primarily for the basic function of cellular immunity and humoral immunity, respectively.7 When pathogens invade, they activate DCs (dendritic cells) by triggering pattern recognition receptors (PRRs) on the cell surface or inside the cell via pathogen-associated molecular patterns

(PAMPs) or danger-associated molecular patterns (DAMPs). This activation results in antigen presentation, up-regulation of co-stimulatory molecules, and polarized cytokines production.8 APCs present foreign antigens to major histocompatibility complexes (MHCs) on cell surfaces forming pMHC (MHC-peptide complex), which are then recognized by T-cell receptors (TCR).9 T lymphocytes are categorized according to the presence of glycol proteins CD4 or CD8, on the cell surface. Naive CD4 T cells are further differentiated into T helper (TH) cells which recognize MHC II ( path 1); while naïve CD8 T cells will differentiate into the cytotoxic T cells (Tc), which recognize major histocompatibility complex I (MHC I)( path 2). In path 1, TH cells recognize MHC II and secrete diverse cytokines that induce qualitatively different types of responses to a wide variety of pathogens. In path 2, MHC I helps Tc cells with differentiation into the effector cytotoxic T lymphocytes (CTLs), which can lyse infected cells10 Previous studies have also indicated that naïve CD4 T cells may further differentiate into subsets, including T helper cells TH1, TH2, TH17, and induced regulatory T-cells (iTregs).11 TH1, TH2, and TH17 cells fight against intracellular pathogens, helminthes, and extracellular bacteria and fungi, respectively.12 ITregs play a critical role in maintaining self-tolerance and modulation of immune responses.13 For example, when viruses and intracellular pathogens invade, DCs produce interleukin-12 (IL-12) to induce the differentiation of CD 4 cells into TH1cells. TH1 cells will produce interferon-gamma (IFN-γ) to eliminate pathogens.14 In addition, TH cells secrete the essential cytokines required to activate Tc cells in Path 2. Except for the effector T cells, CD4 T cells also differentiate into memory T cells (Tm), which respond immediately to eradicate the same pathogens in the second infection. A study by Crotty reported that Follicular helper T cells (TFH cells), a distinct differentiation lineage of CD4 T cells,15 were very important in the differentiation of B cells by producing interleukin 21 (IL-21) and the associated generation of memory B cells.16 B lymphocytes express various cell surface immunoglobulin (Ig)

Michael Kirberger is a Senior Lecturer of Chemistry at Clayton State University. He received his M.S. in Analytical Chemistry and Ph.D. in Biochemistry from Georgia State University. His research interests include protein structure, function and design; biochemical efficacy of natural products; and applications of bioinformatics and proteomics to high-volume datasets.

Wenzhen Liao obtained his Ph.D. degree in Food Science at South China University of Technology in June 2015. He is now a postdoctoral fellow at the University of Saskatchewan. His current research of interest mainly focused on two areas: (1) molecular design, synthesis, modification and biological activity evaluation of bio-nanomaterials. (2) Purification, structure characterization, and bioactivity Wenzhen Liao evaluation (immunoregulatory activity, cytotoxicity, anti-oxidant, anti-hemolysis and anti-inflammatory, etc.) of active compounds in food.

Michael Kirberger

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receptors (B-cell receptors, BCRs) and can immediately recognize specific antigenic epitopes without help from APCs.7 The response of B cells to protein antigens is a highly orchestrated process with participation of both B and T cells. First, specific B cells capture antigens relayed by macrophages, and then encounter T cells which are primed by antigen-presenting DCs. It is at the boundary between T and B cells that the immune response is initiated.17 Subsequently, the T-B cell interactions cause a fast expansion and differentiation of B cells into plasma cells producing pathogen-killing IgM antibodies. Finally, with the help of TH cells, a germinal center is formed. Memory B cells and long-lived plasma cells are generated, and somatically mutated antibodies of different isotypes are secreted with high affinity. In a secondary response, longlived plasma cells render immediate protection while memory B cells differentiate into a burst of antibody producing plasma cells. In addition to the critical role in humoral immunity, B cells also play a major role in immune homeostasis,7 as they function as polarized cytokine-producing effector cells capable of inducing T cell differentiation.18 B cells are also important for the maintenance of normal immune system functions. Abnormalities in B cells may result in autoimmune diseases, immunodeficiency, leukemia, lymphomas and autoimmune diseases.7 Vaccine is a tool for protecting organisms from infections and the chief mechanism of its propylactic treatment is shown in Fig. 1. Vaccine administered into organism containing immunogenic antigen may induce humoral immunity, cellular immunity or both. On the one hand, antigen is recognized by APC and presented to Th cell via MHCII, which activates TH cells. The activated TH cells then activate B cells by cell–cell interactions and secreted cytokines. Actived B cells transform into plasma cells which secrete antibodies in order to scavenge

Jiaoyan Ren is a full-professor in Food Science and Engineering, South China University of Technology (SCUT). She graduated as Ph.D. of Food Science from SCUT and studied in University of Guelph, Canada from 2007 to 2008. She also visited University of Cambridge and Cornell University as visiting professor during 2014–2015. Her research mainly focuses on two areas: (1) Preparation, purification and Jiaoyan Ren identification of bioactive peptides (memory-enhancing pepitdes, anti-aging peptides, antioxidant peptides and anti-cancer peptides, etc.) from food-derived proteins; (2) Structure characterization and bioactivity evaluation (immunoregulatory activity, cytotoxicity, anti-oxidant and antiinflammatory, etc.) of natural compounds ( polysaccharides, polyphenols, etc.) in food.


Review

Fig. 1 The mechanism of vaccination-induced immune responses in human.

pathogen outside the cells as well as memory B cells. On the other hand, antigen is presented to Tc cell via MHCI on the surface of APC and activates Tc together with TH cell which provide help to Tc cells through both cell–cell interactions and the release of cytokines. The activated Tc cells then transformed into CTL cells and memory T cells. CTL cells kill the infected cells and memory T cells are stored. Since the memory B and T cells are capable of staying inside the organism stably and inducing faster immune response in the second infection, organisms obtain immunological memory after vaccination. When the same pathogen invades at the second time, memory B cells fastly transform into plasma cells, which secrete effective antibodies that neutralize pathogens. In the meanwhile, memory T cells transform into CTL cell that kill the infected cells. Both memory B and T cells are protectors for a second invasion of the same pathogen and this is how vaccine works.19 1.2

Advances of vaccine design and their applications

Modern biotechnologies, including recombinant DNA, polysaccharide chemistry, reverse vaccinology, structural vaccinology, and synthetic RNA vaccines, are all methods currently employed in vaccine development.6 More recently, great efforts have been made in the development of subunit vaccines. These differ from inactivated and live-attenuated vaccines in that subunit vaccines do not contain a live component of the virus. These may be produced either by isolating specific proteins directly from the target virus, or sub-cloning the virulent gene into another vector and expressing it into an attenuated bacterium.20 In comparison with traditional vaccines, subunit vaccines significantly reduce toxicity and prevent potential reversion to virulence, and also circumvent many unwanted side effects, including allergies.21 Another outstanding vaccine approach involves immunogenic epitopes. Epitopes are the minimal peptide sequences of an antigen recognized by the immune system, and can be selected for precise direction of immune responses. Vaccines

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based on synthetic peptides of these epitopes can potentially be applied to specific target organs, tissues, cells or intracellular compartments.4 One example is anti-tumour peptide vaccines such as Pharmexa, which targets gastric cancer cell by reflecting factor–receptor 2(TGF R2) and BCL-2 associated X protein (BAX) protein sequences found in MSI tumour cells.22 Another advantage of using these synthetic peptides is that they can be modified with protease-resistant properties to avoid proteolysis in vivo.22 Moreover, in comparison to vaccines targeting whole pathogens or full-length proteins, peptide vaccines are easier to synthesize, exhibit lower rates of mutation, are less prone to contamination by other pathogens, and exhibit higher biostability in vivo. Despite the advantages of subunit vaccines and peptides, several limitations have to be considered. For the subunit vaccine, if the isolated viral protein is denatured or misfolded due to changes in the microenvironment, it may bind to unspecific targets. The immunogenicity of well-defined subunit vaccines is also much lower than that of inactived and live-attenuated vaccines; therefore increasing the duration of the immune response to disease requires more concentrated and more frequent dosages of subunit vaccines. For the synthetic peptides, major drawbacks include poor immunogenic response and degradation of the short synthetic peptide by peptidases due to their unstable tertiary structures. Besides, peptide-based vaccines also present restrictions while being applied to patients of a given tissue type (human leukocyte antigen, HLA, haplotype).22 Therefore, it is necessary to upregulate the immunogenicity of peptide and subunit vaccines. To up-regulate the immunogenicity of subunit vaccines, additional components (i.e., adjuvants) are required to stimulate immune responses. Adjuvants play two important roles. First, adjuvants increase the magnitude of an adaptive response to vaccines based on antibody protection. They can increase not only the response to vaccines with higher mean antibody titer and more rapid sero conversion rates in a population, but also the immune responses produced by elderly and infant vaccine recipients. The inclusion of adjuvants can up-regulate immunogenicity to respond to smaller doses of antigens, which greatly benefits the large scale production of vaccine. Secondly, adjuvants fulfill the need for qualitative alteration of the immune response. Adjuvants are capable of not only speeding up the initial response to an antigen, thus increasing the generation of immunological memory, but they can also produce multiple types of immune response, including humoral, cellular and innate immunity.23–26 What’s more, the breadth, specificity, and affinity of the immune response can be altered by adjuvants.27 In conclusion, the addition of adjuvants in vaccines may enhance and direct immune responses to address specific needs.28,29 Empirically derived adjuvants include Freund’s adjuvant, aluminum salts, oil-inwater emulsions : MF59 and AS03, saponin-based adjuvants, ISCOMs, etc. According to their mechanism of action, and physicochemical properties, adjuvants have also been classified by Coffman28 into five groups based on their targets PRRs: [1] TLR3 and RLR Ligands; [2] TLR4 Ligands; and [3] TLR5

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Ligands [4] TLR7 and TLR8 Ligands; [5] TLR9-CpG-ODN and formulated DNA. Alternatively, adjuvants can be divided into mucosal, parenteral, alum salts and other mineral substances, according to administration route and composition. Currently, multiple adjuvants are being evaluated in pre-clinical trials, but few adjuvants have been licensed for use in human vaccines in either the US or Europe. Adjuvants currently available in vaccines include aluminum salts, oil-in-water emulsions (MF59, AS03 and AF 03), virosomes and AS04.30 Studies of adjuvants will be both promising and challenging, as their anticipated benefits included improved safety, biodegradability, increased stability (i.e., longer shelf life) and improved antigen specificity.31 1.3

main delivery hurdles in the vaccine development

Nanoparticle delivery platforms are promising technologies to improve the efficiency, specificity and sensitivity of vaccines. However, there are several limitations that may hurdles its development. Tatanium dioxide (TiO2) has been considered as a safe and biologically inert material in the use of nanoparticle synthesis. However, in some cases, TiO2 nanoparticle has been shown to induce oxidative stress, resulting in strong immune response, inflammation and cell toxicity.32 The toxicity level depends on the diameter of titanium dioxide nanoparticle, in which smaller than 20 nm in diameter has been shown to cause inflammations in both animal and human.33,34 Cell toxicity of gold nanoparticles were also frequently observed, and the toxicity level greatly depends on size, zeta potential and surface functionalization.35 Usually, molecules less than 12 nm in diameter could cross the blood–brain barrier, while size less than 30 nm could be endocytosed into the cells.36 Gold nanoparticles with sizes of 4, 12 and 18 nm in diameter were found to be nontoxic in human leukemia cell lines;37 however, when diameters goes down to 1.4 nm, the NPs would trigger oxidative stress, mitochondrial damage and necrosis.38 In the case of cationic lipid nanoparticle, the cell toxicity can be caused by a minimum dose for effective therapy. High mortality rates were observed in several cases with systemic administration of synthetic genes or vaccines conjugated cationic lipid particles.39–41 Another concern related to the nanoparticle delivered vaccine is the accumulation of non-degradable or slowly degradable NPs in human body. For example, fluorescent quantum dots were still observed in mice body after 2 years of injection.42 These circulating NPs might potentially interact with non-specific molecules inside of the cells.43 Besides the toxicity and accumulation, instability of the surface antigens on some NPs also hurdles the development of NPs vaccines. Under in vivo condition, the bonds between end group of ligand and inorganic nanoparticles might be fragile due to the dynamic changes of environment. Multiple washing and re-suspending steps may cause damage of the binding or aggregation of several NPs.44 Even though liposome NPs have been extensively used for drug, DNA or vaccine delivery, unstable properties of liposome seriously hampers the improvement of this type of NPs;45 modification of the surface


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with hydrophilic polymer, such as poly(ethylene glycol)-linked phospholipids (PEG-PLs), can reduce the elimination.46,47 Nanoparticle could also be modified or conjugated with glycan or antibody to promote the cross-presentation. To improve the antigens delivery to universal DNGR-1 marker from CD8α + -like DCs, PLGA nanoparticles was conjugated with CLEC9a receptor. Delivery of specific antigens using this nanoparticle increased the cross-presentation capacity of dendric cells.48–51 In another study, an innovated vaccine delivery system was designed by combining αGalactose modified antigens with amphiphilic polyanhydride nanoparticle. This novel vaccine system promoted a higher titer antibody response, germinal center formation, and antibody affinity maturation.52–54 In the development of tumor vaccines, modification of surface of nanoparticles or co-delivery of adjuvants, were used to trigger cross-presentation and CD8+ T cell response. One example is the delivery of ovalbumin (OVA). OVA is an exogenous antigen, proteolysis by proteasomes and amino peptidases of OVA is critical to generate mature antigenic epitopes to induce cross-presentations. By incorporating a cell-penetrating peptide - octaarginine (R8-Lip) into the liposomes, the C-terminal region of the antigen peptide was trimmed and much higher immune response, as well as cross-presentation, were observed.55 1.4

Role of nanoparticles in vaccine development

One of the major challenges for vaccine development is to determine the optimal combination of antigen, adjuvant, delivery system, and vaccine formulation. The delivery system is considered an essential factor for optimizing immunogenicity and bio-tolerability of vaccines, since vaccines prepared using novel technologies, including subunit and synthetic peptide vaccines, present relatively low immunogenicity. Nanomaterial vaccines have been designed to conjugate either synthetic peptides or subunit vaccines into nanoparticles, and have been demonstrated to improve antigen stability, immunogenicity, targeting specificity, and sustained cargo release.56 These vaccines mimic various properties of pathogens, including size, geometry and PAMPs,57 and can function as either prophylactic or therapeutic vaccines, inducing both humoral and cellular immune responses. Vaccine platforms that have been evaluated in recent review include polymeric nanoparticles, liposomes, virus-like particles, peptide amphiphile micelles, peptide nanofibers, and microneedle arrays. Previous studies have reported that nanoparticles can enter living cells of comparable size, thus facilitating the cellular endocytosis mechanism, in particular pinocytosis58,59 and improve the efficacy of vaccine. On the nanoscale, not merely the chemical compositions, but also physical characteristics, and biochemical modifications of materials determine their biological effects in vivo. These are all important parameters that should be considered for the applications of these nanomaterials in vaccines.60 Nanoparticle shape, dose and other factors will also affect the administration effect. Therefore, it is anticipated that nano-


Review

material vaccines with various compositions, sizes, shapes, and surface properties, will be designed to address different requirements.61–63 Moreover, administration routes, including epidermal, dermal, mucosal, intranasal and intranodal administrations, should also be taken into consideration during the design of nanomaterial delivery systems. In this review, we offer an overview of recent advances in nanomaterial vaccines of diverse properties.

2. Polymeric nanoparticles Of all the various nanomaterials, polymeric nanoparticles attract the most attention because they are biologically safe, bio-degradable, and able to protect antigens from degradation.64 A variety of polymeric nanoparticles have been investigated for vaccine development, including poly(lactic-coglycolic) acid (PLGA), polylactic acid (PLA), poly(r-glutamic acid) (r-PGA), and polypropylene sulfide nanoparticles (PPS). 2.1

Poly(lactic-co-glycolic acid) (PLGA)

PLGA is an FDA-approved, biodegradable and bio-compatible polymeric material which has been widely used in the biomedical field. As a proven delivery platform for a variety of agents, PLGA nanoparticles are excellent carrier systems for both antigen and adjuvant. This platform has been extensively applied in the delivery of small molecular drugs, and utilized for the determination of immunity types. However, potential limitations include possible denaturation of proteins during particle formation and low encapsulation dosage. A related study by Moon et al. reported the fabrication of a pathogen-mimicking polymeric vaccine (VMP001-NPs) composed of a lipid-enveloped PLGA (Fig. 2a),65 a candidate malaria antigen (VMP001), and an immunostimulatory molecule monophosphory llipid A (MPLA). The VMP001 was conjugated to PLGA incorporated with MPLA. Results of this study indicated that vaccination with VMP001-NPs promotes germinal center formation, which significantly increases the durability of the antigen-specific antibodies titer and more efficiently balances T1/T2 responses in vivo in comparison with soluble protein-derived vaccines.65 Another successful application of PLGA utilized a combination of intranodal injection and controlled release materials, and it was recently reported that intra-lymph node (i.LN) injection enhances the potency of DNA, RNA, peptide, protein and dendritic cell-based vaccines.66 In a related study, Toll-like receptor-3 ligand poly (inosinic : cytidylic acid)( polyIC)was encapsulated into biodegradable PLGA for intranodal immunization injection, and this reportedly produced a measurable increase in the persistence of polyIC, accumulation of Toll-like receptor agonists in lymph nodes (LNs), and increasing duration of the activated dendritic cells.67 Results of these studies suggest the potential for much broader applications of intralymph node immunization with slow release-formulated adjuvants as a promising method for therapeutic and prophylactic vaccine development.

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Fig. 2 (a) Lipid-enveloped PLGA nanoparticles. Lipid-enveloped PLGA nanoparticles are comprised of a polymer PLGA core covered with a lipid membrane. By emulsifying an organic phase of polymer PLGA and co-dissolved lipids in water, PLGA NPs are self-assembled with lipid coatings surrounding each particle. On the particle surface, the lipid bilayers can be combined with lipophilic pathogen-associated molecular patterns (PAMPs) and conjugated protein antigens. Particles are then subsequently PEGlated, forming the final composite. (b) PEG17-blPPS30. Polymersomes are derived from the self-assembly of a poly (ethylene glycol)(PEG)-bl-poly( propylene sulfide)(PPS) block copolymer amphiphile. Since PEG17 is hydrophilic while PPS30 is hydrophobic, the combined structure forms a monomer that self-assembles into polymersomes in aqueous solutions, and these structures resemble a phospholipid bilayer. Nanoscale polymersomes in different sizes are obtained by extrusion through various nanoporous membranes. The aqueous interior is responsible for transporting hydrophilic payloads (e.g., antigens or adjuvants lacking the need for chemical modification), while the lyotropic membrane wall is used to retain hydrophobic molecules(other adjuvants).

Engman et al. formulated ovalbumin and anti-sense oligonucleotides into PLGA nanoparticles as type 1 diabetes vaccines.68 After being internalized by dendritic cells, anti-sense oligonucleotides would silence the expression of co-stimulatory molecules and therefore inactivate the dendritic cells. Upon migration to pancreatic lymph nodes, these inactivated dendritic cells would induce the expansion of antigen specific Foxp3+ regulatory T cells for the reverse of diabetes. More importantly, it was demonstrated that antigens in the microsphere was not required since antigens may be supplied in draining lymph nodes. This finding is of great importance in vaccine design because the current paradigm considers antigen has to be provided. 2.2

polypropylene sulfide (PPS)nanoparticles

PPS nanoparticles have also been reported in a number of new applications, including: antigen and/or adjuvant delivery; as well as intranasal, mucosal or pulmonary administration. A study by Scott et al. reported the use of oxidation-sensitive nanoscale polymersomes – PEG-bl-PPS (Fig. 2b)64 to deliver both antigen and adjuvant to dendritic cell (DC) endosomes.64 The calcein-loaded polymersomes used in the study were observed to release their payloads in multiple DC endosome

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compartments, and later, in the cytosol. Oxidation-sensitive polymersomes can function as vaccine carriers capable of inducing cell-mediated antigen-specific immune response by using the Toll-like receptor agonists gardiquimod, R848, and ovalbumin, as payloads.64 A study by Thomas, et al. reported efforts to determine whether targeted delivery of adjuvant to the tumor-draining lymph node (TDLN), which might already be bathed in tumor antigen, could promote anti-tumor immunity and slow tumor growth rate.69 In this study, the adjuvants CpG- or paclitaxel-incorporated Pluronic-stabilized PPS (CpG-NP and PXL-NP) with average diameters of 30 nm, were administered to effectively target and induce DC (CD11c+) activation within LN soon after intradermal administration. Results of this study indicated that the anti-tumor immune response was modified by delivery of CpG to TDLN.69 Previous studies have also demonstrated that subunit vaccines and high adjuvant doses are being evaluated for their abilities to induce robust CD8+ T-cell immunity. A subsequent study by Titta et al. demonstrated high adjuvant doses were not required to further enhance immunogenicity. They separate ultra-small polymeric nanoparticles co-delivering antigens and adjuvants, and inherently encapsulating the similar size cargos with themselves. Moreover, further research has concluded that coupling CpG-B or CpG-C oligonucleotides to NPs provides improved dual-targeting of adjuvants and antigens.70 In a related study, Ballester et al. reported development of a new vaccine delivery system capable of inducing robust T-cell immunity against Mycobacterium tuberculosis (Mtb).71 This delivery platform is comprised of 30 nm CpG-adjuvanted NPs conjugated with tuberculosis antigen Ag85B(NP-Ag85B), which target lymphoid tissues based on size of the conjugates. Following administration by both pulmonary and mucosal routes in mice, both the NP-based delivery system and pulmonary route have been shown to be very efficient for efficacious TB vaccination development. Cross-presentation of exogeneous antigens was observed, which is a key element required for activation of cytotoxic T cells (Tc). A study by Hirosue et al. reported the use of a synthetic nanoparticle vaccine platform with disulfide conjugated ovalbumin (OVA) peptides to investigate the relevance of the delivery platform and antigen conjugation scheme. Results of this study indicated that reductionsensitive conjugation of MHC1 and OVA peptide produces better antigen cross-presentation than non-reducible linkages.72 Additionally, a study by Stano et al. reported that the antigen OVA was most effectively delivered into both MHC I and MHC II presentation pathways by NPs ranging from 30 to 200 nm. Moreover, the 200 nm NPs are special nanocarriers for prophylactic vaccines against mucosal pathogens because they require protection from mulitifunctional CD4+ T cells. Additionally, this research provided new insight into how the size of an antigen-conjugated nanoparticle determines the immune response type against protein antigens.73 Stano et al. provided an example of a different NPs delivery system using 50 nm PPS conjugated with thiolated antigen and adjuvant proteins via reversible disulfide bonds. Using this approach for the application of mucosal vaccination, OVA,


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the antigens induced cytotoxic T lymphocytic response in both lung and spleen tissues, accompanied with humoral responses in the airways. Conversely, the adjuvant TLR5 ligand flagellin, further enhance humoral response in the airways and cellular response with a TH 1 bias.74 Finally, a study by Tzu-Yu’s reported the fabrication of a delivery system based on a polyacrylate polymer conjugated with a peptide epitope derived from HPV-16E7 protein for vaccine delivery. Results of this study demonstrated that this poly-specific NP could efficiently deliver the protein epitopes to cervical cancer cells.106

3

Liposomes

Liopsomes and liposome-derived nanovesicles also play important roles in vaccine development. Liposomes are spherical vesicles with at least one lipid bilayer surrounding an aqueous core (Fig. 3).79 In liposomes, the antigen can be in the core of the liposome, buried within the lipid bilayer or incorporated in the liposomal surface. Liposomes deliver not only drugs, but also genes, dyes and textiles, and is particularly useful for broad biomedical applications due to their potent immunogenicity, and manufacturability.75,76 Liposomal delivery of amphotericin B and doxorubicin are two of the most important successful nanodelivery systems.77 Lipid vesicles can be classified as either unilamellar, multilamellar, or polymerized types. Due to their versatility and plasticity, lipid vesicles can be fabricated with desired features, including: chemical components, charge, size, entrapment capabilities, and location of antigens and/or adjuvants.78 Cellular uptake of lipid vesicles is determined by their diameter/polydispersity, while colloidal stability depends on the numbers of bilayers. The stability of liposomes plays a key role in the potency of lipid vesicles;79 however, efforts thus far to develop effective stabilizing strat-

Fig. 3 Liposomes. Liposomes are interbilayer-crosslinked multilamellar vesicles (ICMVs) formed by stabilizing multilamellar vesicles with short covalent crosslinks, linking lipid headgroups across the opposing faces of adjacent tightly stacked bilayers within the vesicles walls. (1) Preparation of anionic maleimide-functionalized liposomes containing antigen and adjuvants. (2) Divalent cations are added to help form the MLVs. (3) Membrane-permeable dithiols are added to form the crosslink between apposed lipid bilayers in the vesicle walls. (4) PEG-SH are added to conjugate particles with thiol-terminated PEG antigens are carried in the aqueous core and adjuvants are embedded in the vesicle walls.


Review

egies that can be achieved under mild conditions have met with little success.80 To investigate the impacts of structure and composition of liposomes on strength and durability of humoral and helper T-cell responses in mice, Melissa Hanson et al.81 incorporated membrane-proximal external region (MPER) peptides onto the surface of high-TM liposomal vesicles, and adjuvants, such as monophosphoryl, were conjugated within the lipid bilayer. Results of this study indicated that the conjugated nanoparticle significantly enhanced the MPER-specific antibody production and cellular immune response.81 Similarly, Irvine et al.82 reported the development of interbilayer-cross-linked multilamellar lipid vesicles (ICMVs) as a new class of lipid drug carriers. This nanoparticle is composed of stabilizing multimalellar vesicles with short covalent crosslinks linking the lipid head groups across the opposing faces of adjuvant tightly stacked bilayers within the vesicles wall.82 Compared with multilamellar vesicles of the same lipid composition, ICMVs are capable of encapsulating high levels of protein, retaining them in situ within skin, and controlling the release of protein at an extremely slow rate. In addition, ICMVs can be quickly degraded by lipases which are normally present in high concentrations in cells.83 One example is the loading of model protein antigens (e.g., OVA) and lipid-like immunostimulatory particles into ICMVs, which has been observed to efficiently stimulate an antigen specific immune response.84 In another study, Moon et al.79 reported that ICMVs, loaded with Plasmodium vivax circumsporozoite antigen (VMP001), are capable of inducing a durable antibody response at low dosage. Additionally, increased breadth and avidity of humoral responses, and enhanced antigen delivery and Germinal Center Formation (GC), were also observed.79 ICMVs have also been adapted for the delivery of recombinant subunit vaccines, for the purpose of generating stronger humoral and cellular immune responses.82 Results of another study made by Li and his colleagues85 reported that the combination of two TLR agonists, polyinosinic–polycytidylic acid ( polyl : C) and monophosphoryl into protein-loaded ICMVs was safely applied for pulmonary immunization of mice via subcutaneous vaccination, and subsequently produced an increase in the antigen transported to draining lymph nodes. Results from these experiments demonstrated that the application of ICMV primed 13-fold more cells than soluble vaccines. Furthermore, ICMV also induced increased expression of mucosal homing integrin a4b7+ and generated long-lived T cells in the lungs and distal mucosa, which is strongly biased towards production of effector memory T cells (TEM). In summary, this particle was observed to target APC-rich pulmonary mucosa, leading to robust T cell responses in order to protect mucosal surfaces.85 The ultilization of liposomes as delivery system also brings one of the greatest pharmaceutical challenges in vaccinology that the antigens encapsulated by liposomes are delivered to cytosol of antigen-presenting cells (APCs) which successfully

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Review

stimulate MHC I-restricted CD8+ T-cell responses instead of MHC II presenting path in current vaccines and later act on intracellular infections or cancer. Taking the benefit of that, Hjalmsdottir A. et al.86 construct method to develop photosensitive particulate vaccines that stimulate strong CD8+ T-cell responses. In their research, the photosensitiser TPCS2a was formulated in liposomes which elicits strong CD8+ T-cell responses through MHC II processing and presentation path. Another potential application is the BLP25 liposome vaccine (L-BLP 25) used for lung cancer therapeutics. This vaccine has been introduced into clinical trials for patients with unresectable, stage 3 non-small cell lung cancer, and has been shown to prolong the survival times of patients, especially those with stage 3 Blocoregional disease.87

4 Virus-like particles Another method for delivering peptide antigens is to use noninfectious, non-replicating virus-like particles (VLP). VLPs are composed of biocompatible self-assembling capsid proteins that lack infectious nucleic acid sequences.88,89 VLPs are considered ideal nano vaccine systems since they utilize the evolved viral structure, which is opitimized by the nature. VLPs are capable of displaying multiple copies of antigens across the surface of the particles, but T-helper-cell epitopes and pathogen-associated molecular patterns are also present. Pathogen-associated molecular patterns can be recognized by the innate immune system, which enhances the immune response.90 VLPs can be made from an artificial recombinant viral envelope or self-assembled capsid proteins of 20–100 nm diameter capable of presenting multiple antigenic peptides on their surface. Capsid proteins are derived from a parental virus, genetic insertion, fusion of foreign antigenic epitopes, or the chemical conjugation with pre-assembled VLPs.21 Due to their naturally-optimized nanoparticle size, as well as their repetitive structural order, VLPs have the potential to induce potent immune responses independently of adjuvants.91,92 Studies have reported that TLR, an exceptional adjuvant, can improve the effectiveness of certain VLPs (92). At present, only a limited number of virus-like particle vaccines have reached market, including recombinant hepatitis virus B vaccine and the human papiloma virus vaccine. Nevertheless, there are still some pathogens that remain challenges for VLP vaccine development, including pathogens that directly infect immune cells, evade the immune system, or display rapid genetic drift. Two of these virus-like nanoparticles were selected for further discussion in this review, as detailed below. 4.1

Self-assembling polypeptide nanoparticles (SAPNs)

Self-assembling polypeptide nanoparticles (SAPNs) represent a novel vaccine platform comprised of linear peptide (LP) monomers self-assembled into either icosahedral or octahedral symmetry (Fig. 4).90,93,94,112 SAPNs are capable of repetitively

Biomater. Sci.


Fig. 4 (a) SPAN (self-assembling polypeptide nanoparticles) the linear peptide (LP) monomer is designed to address a specific need, including the design of its basic structure and an extended conformation presenting the specific epitopes. It is comprised of two coiled coils connected by a short linker region which induces self-assembling of the monomers into spherical nanoparticles with either icosahedral or octahedral symmetry. SPAN is formed by the oligomerization of 3- and 5-stranded coiled–coiled domains within the momomer. Specific repetitive epitopes from antigens or B/T cells are presented on the surface of SPAN. (b) SVLP (synthetic virus-like particles). SVLP comes from the selfassembly of artificial, synthetically-derived building blocks which form particles similar in size and composition to natural viruses. These contain proteins and lipids, but not nucleic acids. Antigens are first recognized and assembled, connecting to the head of protein of the artificial synthetically derived lipopeptide building blocks. According to the principles of protein–protein and lipid–lipid recognition, the coiled coils of peptide chains and lipid tails oligomerize, resulting inself-assembly into the SVLP, a spherical-like particles with a protein cover and a lipid core. (c) Peptide amphiphiles micelles. Micelles are dynamic structures in which individual monomers composed of peptide epitope and lipid tail can escape and relocate to cell membranes or other micelles. Monomers comprising the peptide amphiphiles micelles are lipopeptides artificially synthesized with lipid tails and protein heads containing specific epitopes. Monomers self-assembled into cylindrical Peptide amphiphiles micelles display a high density of peptide on the surface.

displaying antigenic epitopes, and research suggests they represent a viable method for inducing robust and long-lived adaptive host immune response in mice.93 A number of studies have focused on constructing virus-like SAPNs. Sankhiros et al. reported immunization of specific pathogen free (SPF) chickens with two novel vaccines, M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. The chickens were infected with 107.2 EID50 of H5N2 low pathogenicity avian inflenenza (LPAI) virus. Results of this study indicated that the Tetra-M2e with adjuvant provided significant protection against the LPAI virus, which was seen as reduction of the AI virus shedding, and that self-assembling polypeptide nanoparticles exhibit great promise as a platform for AI virus vaccine development. Additionally, in a very important study by Babapoor et al., a prototypic malaria vaccine was fabricated based on a highly versatile self-assembling polypeptide nanoparticle (SAPN).94 The vaccine contained the B cell immunodominant epitope with a tandem repeat sequence (DPPPPNPN), generated from the circumsporozoite protein of Plasmodium bergheri.


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Similarly, the SAPN construct P4c-Mal vaccine reportedly induced a long-lived, protective immune response, where the SAPN platform functioned as both a delivery system and as an immunological carrier.93 To better understand the mechanisms behind these observed results, McCoy et al.95 used SAPN vaccine presenting CSP epitope (PfCSP-SAPN) to induce epitope-specific antibody titers, in order to target P. falciparum sporozoites through the classical pathway of complement, as well as through long-lived and effector memory CD8+ T-cells. In a related study,96 the administration of SAPN used to present both CD8+ T cell epitopes and CD4+ epitopes induced long-lived antibodies and cell-mediated immune response to pfCSP, and prevented infection in mice.96 Similarly, results of a study by Bissati et al. also indicated that SAPN may provide a useful platform for vaccine design to fight against toxoplasmosis.97 4.2

synthetic virus-like particles (SVLPs)

Self-assembling building blocks can be utilized to produce synthetic virus-like particles (SVLPs).This type of nanoparticle can be arranged to emulate the size and composition of natural viruses, proteins or lipids (Fig. 4a),94 and combine the advantages of naturally derived VLPs, with the potential for further chemical modification and optimization. SVLPs may be assembled with a lipid core inside, following the principles of protein–protein and lipid-lipid recognition.98 A recent study using SVLPs reported that the size of some small virus capsids could be reproduced in significantly homogeneous form by starting from a designed self-assembling coiled-coil lipopeptide building block. The SVLP was further engineered by adding artificial T-helper-cell epitopes and Toll-like receptor ligands. Results of the study demonstrated that multiple copies of antigen can be loaded onto the surface of the particles, and that the antigen-loaded particles induced adjuvantfree stimulation of antigen-specific humoral immune responses.98 Similarly, Arin et al. fabricated nanoparticles loaded with promiscuous T-helper epitope and synthetic B-cell epitope to mimic the effects of circumsporozoite protein of Plasmodium falciparum, which was later shown to elicit robust humoral immune responses in mice and rabbits. Additionally, these particles also elicited the production of antibodies capable of cross-reacting with the parasite, thus revealing their potential use in synthetic vaccine design.99 In arelated study to determine how SVLP induced an adaptive immune response without adjuvants, Sharma et al. concluded that DC potentially uses multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raftmediated micropinocytosis.100 In Thrane S ’s research, placental malaria VLP vaccine formulated by mSA-VAR2CSA antigen encapsulated on the Avi-L1 VLP platform is demonstrated to be much higher efficacious than two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA in C57BL/6 mice. A VLP vaccine display platform is developed by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTagTM) has been inserted and later monovalent streptavidin (mSA)-


Review

fused proteins are anchored to the biotin, displaying a dense and repetitive VLP-display of the vaccine antigen. The mSAVAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine while the VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, which demonstrates that the Avi-L1 VLP-platform may facilitate optimal VLPdisplay of large and complex vaccine antigens.101

5

Peptide amphiphiles

The major problems associated with subunit vaccine design include limited immunogenicity, poor surface accessibility, and conformational stability. Peptide amphiphiles (Fig. 4b),90 which are peptide-based molecules comprised of both hydrophilic and hydrophobic domains, represent a solution to these problems, as the spontaneous formation of micelles will not denature or compromise the bioactivity of peptides, while the α-helical conformation of the peptides can maintain the stability of the molecule. In addition, the nano-size of the micelles enables passive targeting of peptides to the desired site of action through leaky vasculature present at tumor and inflamed tissues. Thus, peptides in phospholipid micelles present high efficacy and stability, with reduced doses required to induce the immune response, and several studies have demonstrated that phospholipid micelles are safe, stable and effective delivery options for peptide drugs.102,105,109 Another recent study has reported that alipoamino acidbased carrier is capable of inducing strong immune response when it is covalently linked to a peptide epitope.103,104 In a study by Azmi, the peptide epitope (J14) was covalently linked to lipoamino acids as an immune-stimulant carrier, which resulted in improved vaccine immunogenicity.105 Similarly, in a study by Karen,107 a vaccine antigen was fabricated using a lipid-core peptide (LCP), modified to include adjuvant properties, and tested using mouse models. Results of the study indicated that the modified LCP was able to activate antigenpresenting cells in vitro, and Tc responses in vivo, and stimulate the development of a protective anti-tumor immune response.107 Similarly, Zaman et al.104 reported that lipopeptidic nanoparticles can be greatly influenced by the combined effect of epitope and molecular size while inducing an immune response.104 Moyle and his colleagues108 designed a novel vaccine composed of a synthetic gene incorporating seven streptococcus M protein strain-specific antigens and J14, attached to an LCP, which was demonstrated to be an efficient vaccination in a murine model system, without the requirement for adjuvants.108 Zaman et al.109 reported the design of a vaccine capable of inducing optimal antibody response. This vaccine featured a C-terminal lipid moiety with a 16 carbon alkyl chain, P25 located at the N-terminus, and J14 attached to the side chain of a central lysine residue.109 In a similar experiment, Simerska et al. conjugated OVA257-264 from OT 1(CD8+) and OVA323-339 from OT 2(CD4+ specific) peptides to LCP.

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Following administration of the conjugates in mice, results indicated that the LCP-ovalbumin vaccine system was capable of stimulating a robust antigen-specific CD8+ T cell response.103 A study by Black et al. reported that the assembly of peptide amphiphiles (PAs) into nanometer-sized micelles can impart self-adjuvanting properties. This makes PAs very useful as peptide antigen delivery systems, asmicelles from PAs not only effectively deliver the antigen, but they can also offer a concentrated multivalent display of peptides, and can be used as synthetic self-adjuvanting vaccines that stimulate receptors such as toll-like receptors(TLRs) on DCs.110,112,122 In Vincenzi M’s research, amphiphilic peptides are synthesized which are consist of lipophilic derivatives of PepE and PepK bearing two stearic alkyl chains and/or an ethoxylic spacer.111 PepE and PepK Recently, are two linear ester peptides provided with the following sequences: Y-G-E-C-P-CK-OAllyl (PepK) and Y-G-E-C-P-C-E-OAllyl (PepE),both of which contain the “CPC” motif, the binding motif for the CXCR4 receptor which represents a well-known target for cancer therapies. These peptide amphiphiles aggregates stably presenting conformational features that are typical of intrinsically disordered molecules. They can be used for investigate the molecular recognition processes involving the CXCR4 receptor in vitro cell-based assays.

6 Peptide nanofibers Self-assembled nanofibers are regarded as a promising hydrophobic drug delivery system for clinical treatment of different diseases,113 especially cancer, and are also proving to be useful in biomedical and biotechnological applications like tissue engineering, wound healing, cell delivery, enzyme catalysis, biosensors and electronics.114–118 Peptide nanofibers presenting peptide epitopes represent another example of a novel vaccine. Generally, peptide nanofibers (Fig. 4c)112 conjugated with short peptide epitopes function not only as the delivery system, but they may also incorporate adjuvants that can stimulate robust and long-lived antibody responses.63 In Another study, Hudalla and his colleagues119 describe a strategy that employs ‘β tail’ tags which are induced to coassemble into nanomaterials when mixed with additional β-sheet fibrillizing peptides shown in Fig. 5.120 This is an effective method to assemble nanofibers with exceptional compositional control, and allows for the insertion of different kinds of protein into the peptide nanofibers. This is finally illustrated by achieving precisely targeted hues with the help of a mixture of fluorescent proteins.119 A study by Rudra et al. reported that OVA-Q11 helped elicit strong T cell-dependent antibody responses lasting for a long time (at least one year), and that nanofibers of OVA-KFE8 elicited a similar robust antibody response, whereas the self-assembling peptide KFE8, did not.114 Pompano et al. reported that the modular assembly of nanofibersis able to tune T epitope doses within non-inflammatory material-based vaccines, induce strong humoral and

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Fig. 5 (a) Peptide nanofibers. (1) Fibril morphology and secondary structure are formed when Q11 conjugate with T/B cells epitope, forming integrated nanofibers. (b) Protein nanofiber. A schematic representation of fusion proteins containing a β-sheet fibrillizing domain integrated into Q11 nanofibers. The initial monomer consists of Q11 and the epitope protein ligands. These peptide nanoparticles are serving as binding sites for target protein cutinase-GFP fusion proteins. The Q11 domain then assembles into fibrillar aggregates, presenting epitopes on the surfaces at the end of a flexible spacer, Finally, the epitopes and the protein ligands are precisely combined and attached the target proteins.

cellular immunity, and specify the resulting CD4 effector types.120 Additionally, a study by Chen et al.121 comparing alum and complete Freund’s adjuvant, reported that selfassembled β sheet-rich peptide nanofibers are capable of inducing stronger antibody responses with no measurable inflammation in vivo. Epitope-bearing nanofibers may elicit antigenspecific differentiation of T cells into T follicular helper cells, B cells, and neutralizing antibody IgG with high-titer, and high-affinity against influenza in vitro. As a result, selfassembled peptide nanofibers may be a good device for noninflammatory vaccines design,121 and yet with desirable thermostability.132 Fiber is the most common type of shape for peptide-based nanomaterials, while peptide nanofiber displays outstanding property for enhancing antigen-specific humoral and cellular immune responses.60

7 Microneedle arrays Skin is an ideal target for vaccine administration because of the inherently robust immune response to invading pathogens, the dense matrix of resident innate immune cells, and the high frequency of epidermal and dermal APCs.123–125 In contrast to traditional methods, transcutaneous administration offers a pain-free, -invasive, safe, and convenient approach for therapeutic delivery. This approach may also greatly reduce the generation of dangerous medical waste and diseases derived from needle-reuse and needle-based injury. Additionally, transcutaneous administration has the potential to improve the potency of immunity induced by vaccines, and


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enhance the clinical effectiveness of treatment, by providing more efficient delivery of drugs which are susceptible to firstclass metabolism in the liver. More importantly, since there is no need for refrigeration during transportation, transcutaneous vaccines are much easier and cheaper to distribute, even for the environmentally sensitive biological vaccines.126 A recent development in this area, microneedle arrays, represents a safe and convenient transcutaneous delivery system for vaccines. This approach is widely employed for delivering diverse bioactive materials, including molecular weight hydrophilic biologics, such as whole proteins, by painless mechanical disruption of the stratum corneum.126 This type of research initially focused on the construction if single-path microneedle arrays capable of providing rapid release or controlled release of therapeutic formulations. For example, bolus is known as a typical rapid releasing drug. Strategies for obtaining sustained drug release with microneedle arrays include employed intradermal injection from hollow microneedles,126 delivery from coatings on microneedle surfaces, and encapsulating drugs directly into the biodegradable microneedles, such as PLGA microneedles.127,128 DeMuth et al. reported the development of a PLGA microneedle array coated with PEMs( polyelectrolyte multilayers) incorporated by ICMV (Fig. 6).126 Microneedles coated with PLGA nanoparticleloaded PEMs were found to be capable of rapid implantation of particle-loaded films in skin, and lipid nanocapsules were able to provide sustained drug release to the skin.128 However, one problem associated with this approach involved determining how to insert the intact nanoscale vesicles into the erodible PEM system, since they ruptured spontaneously when assembled, or when drying the resulting films. Prior to the development of multilayer assembly, the problem could be

Fig. 6 PLGA/ICMVs Microneedle array. The PLGA microneedle arraysare coated with multilayer films, which are assembled from biodegradable cationic poly(b-amino ester)(PBAE) and negatively-charged ICMVs. Antigens and adjuvants are loaded into the ICMVs. After application of the microneedle in the skin, the poly-1/ICMV)PEMs are transferred into the skin, followed by hydrolytic degradation of poly-1 which leads to the disintegration of PEM into fractions. ICMVs then release the payloads into the tissues or APCs.


Review

solved by creating robust vesicles via in situ silica polymerization or polyelectrolyte adsorption on vesicles surface,129 however DeMuth et al. reported a new approach that also enhanced the stabilization of vesicles by introducing covalent cross-links between adjuvant phospholipid bilayers in the wall of vesicles.126,129 Using this approach, the fabricated microneedle systems were used to deliver a protein vaccine formulation to mice. The transcutaneous drug delivery system was built using PLGA microneedle arrays, which were coated with degradable PEM films through layer-by-layer assembly of a biodegradable cationic poly(b-amino ester)(PBAE) and negatively charged ICMVs. A protein and molecular adjuvant, monophosphoryl lipid A, western loaded onto the ICMVs. Films were transferred from the PLGA microneedle array to the cutaneous tissue of the mice within 5 min of application, where they remained in situ. The ICMV cargos slowly dispersed from the multilayers to the treated tissue in vivo in the subsequent 24 h period. The lipid nanocapsules taken up by the tissues released antigens that triggered the activation of APCs. Results of this study indicated that this approach shows great potential to mediate effective transcutaneous vaccine delivery, combined with slow, sustained protein release and enhanced humoral immune response. A related approach involves the construction of a two-path microneedle array, which combines a rapid release phase and a sustained release phase. To avoid potential infection caused by this barrier-disrupting system, therapeutics should be rapidly delivered into the skin through a brief application of the microneedle array. However, rapid drug release can lead to rapid clearance, therefore large concentrations of drugs are required to maintain a therapeutic dose. Conversely, since sustained drug release is essential for many therapeutics or specific mechanisms of immunity, prolonged application time is also required. To overcome these limitations, DeMuth et al. developed a two-path microneedle array that was capable of balancing duration and kinetics of exposure to antigen and adjuvant. This novel microneedle array was comprised of drugloaded PLGA microparticles or solid PLGA tips with a supporting, and rapidly water-soluble, poly(acrylic acid)(PAA) matrix (Fig. 7a).126 In the mouse models, the microneedle patch perforates the stratum corneum and epidermis. After penetration of the outer skin layer, the PAA binder rapidly dissolves within the interstitial fluid of the epidermis. Subsequently, the microparticles or solid polymer microneedles were implanted in the tissue and were retained there after removal of the patch. For weeks, these polymer deposits remained in the skin, and continued to release their encapsulated cargos very slowly, thereby delivering a very low dose. Results of this experiment indicated that the synthesized microneedles delivered the subunit vaccine, and induced arobust cellular immune response. Furthermore, vaccination utilizing PLGA/PAA composite microneedles has been shown to exhibit improved proliferation, stronger antigen-dependent cytokine secretion, and changed memory phenotype in the context of CD8 T cell immunity. The DeMuth lab subsequently designed a silk/PAA composite microneedle array comprised of a silk tip supported by a PAA

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Fig. 7 (a) PLGA/PAA Microneedle arrays. Two paths exist because of the different manufacture methods. Path 1: after the PDMS mold forms, it is first filled with PLGA by centrifugation. The PLGA MP is dried to form the solid bulk. Concentrated PAA solution is used to fill the surface of PDMS molds, which forms the supportive matrix. After drying, microneedles form and are removed from the molds. Path 2: after PDMS mold forms, it is first filled with PLGA fused at high temperature, and then chilled to form the solid tip. Concentrated PAA solution is used to fill the surface of PDMS molds which forms the pedestal. After drying the PAA, microneedles form and are removed from the molds. (b) Silk/PAA Microneedle array. Silk/PAA PDMS molds are made by laser ablation and treated by plasma. Silk-vaccine formulation is added in the molds and centrifuged in order to fill the cavities. Hardened tips are formed after drying the silk. Vaccine-loaded PAA is the nadded to the mold, and the mold is centrifuged to fill the cavities. After drying the PAA, arrays form and are removed.

base (Fig. 7b).130 This array functions through two distinct pathways, inducing both rapid release and slow sustained release of therapeutics. In practice, the microneedle patch was briefly applied to skin, and the PAA bases dissolved rapidly to deliver a protein subunit vaccine bolus. At the same time, persistent silk hydrogel depots, designed to slowly release the vaccine formulation over a period of 1–2 weeks, were implanted into the skin. Silk hydrogels create implantable hydrogel microneedle matrices for both whole protein and highly tailorable vaccine release. It also makes loading of biomolecules an efficient single step process. Additionally, this approach exhibits the potential to generate cellular and

Table 1

humoral immune responses. Due to the fact that microneedles inserted into murine need to be stored at room temperature, it also successfully avoids expanse of cold chain transportation.130 In Maaden’s research, pH-sensitive microneedle arrays are coated with 10 layers of inactivated polio vaccine (IPV) particles and N-trimethyl chitosan chloride (TMC) via electrostatic interactions, forming the desired vaccine.131 After topical administration in mice, immunogenicity of the vaccine is detected. It is demonstrated that vaccination inductes IPV specific antibody responses, illustrating that the vaccine is practically applicable. According to this research, topical administration of pH-sensitive microneedles coated with polyelectrolyte multinanolayers composing of antigens and oppositely charged polymers is demonstrated to be a functional approach for microneedle-based vaccination.

8 Conclusion The purpose of this review was to provide a summary of several typical types of nanomaterials recently developed for vaccine delivery. There is no doubt that novel vaccines are in great demand due to both the reality and the potential threat of epidemics. To combat these emerging infections, chronic diseases, cancers or autoimmune diseases, more elaborate studies of vaccinology, immunology and bio-technologies are required. This review focused primarily on six vaccine platforms, including polymeric nanoparticles, liposomes, virus like particles, peptide amphiphiles micelles, peptide nanofibers and microneedle arrays. Each vaccine delivery platform exhibited strengths and weaknesses, indicating that further research will be required to improve these systems. The most significant challenge for improved vaccine development is the capability of inducing an extended protective immune response, which may be dependent upon a better understanding of the routes of infection. Immune responses have been improved by the

Examples of nanoparticle based vaccines that have been applied in clinical test

Clinical tested NPs vaccines

Vector components

Antigen

Evaluation

Hepatitis B virus

PLA, PLGA

Human papilloma virus Malaria

Virus like particle (bacterialphage) Inorganic/lipid

Hepatitis B surface antigen Capsid protein L1 + L2 rMSP1

Streptococcus pneumoniae HIV-1

Liposome gold

Polysaccharides, OVA

r-PGA

Gp120

Liposome

Lung cancer

NPs > 500 nm in diameter induces stronger and more specific immune response than smaller NPs133,134 Immunization with hybrid PP7 VLPs displaying 18/1 L2 showed higher antibody responses against HPV1 L2135 Iron Oxide conjugated nanoparticle (rMSP1-IO) shows significant induction of parasite inhibitory antibodies, highly stability and very now systemic toxicity136 Glyconanoparticles containing 45% of tetrasaccharide and 5% OVA (323–339) triggered specific anti-Pn14PS IgG antibodies.137 gamma-PGA NPs were found to be a much stronger inducer of antigenspecific CD8+ T-cell responses than non-biodegradable polystyrene NPs.138 A peptide vaccine strategy targeting the exposed core peptide of MUC1. L-BLP25 induced a cellular immune response characterized by T-cell proliferation in response to MUC1 and production of IFN-γ.139

BLP25 liposome vaccine

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use of antigens, adjuvants, and the delivery systems, used to treat emerging infectious diseases. Cell-mediated immune responses are still unachievable for many other types of diseases, such as tumors, HIV, malaria, tuberculosis, and hepatitis C infections, among others. The information provided in this review was intended to summarize the current state of development in this emerging area of research, and to suggest potential new approaches to addressing the problems associated with advanced vaccine design. Nanomaterial vaccines have brought new hopes for human beings with diseases that lack efficacious vaccines, including allergy, cancer, inflammatory disease and infections. Compared with traditional vaccines such as attenuated and inactivated vaccine, vaccines formulated by nanomaterials are always considered to be much safer for avoiding introducing the whole pathogen into organism, which might cause the reversion of virulence. Instead, nanomaterial vaccines elicit immune response via artificial-designed antigen with high immunogenicity and limited toxicity. Up to now, many clinical relevant nanomaterial vaccines have been introduced into clinical trials. Several examples of nanoparticle based vaccines that have been applied in clinical test are shown in Table 1. Proved by a few animal studies and studies of immune cells, nanomaterials may exacerbate an underlying allergic condition through increases in circulating IgE levels.140 With these unexpected side effects solved in the near future, nanomaterial vaccines are hopeful to be widely utilized in clinical treatment and prophylactic immunization.

Conflict of Interest All authors declare no conflict of interest.

Acknowledgements This study was supported by the Guangdong Natural Science Funds for Distinguished Young Scholars (no. S2013050013954), Program for New Century Excellent Talents in University (NCET-13-0213), Guangdong Special Funding for Outstanding Young Scholars (2014TQ01N645) and Guangdong Science and Technology Planning Project (2015A010107003).

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