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ScienceDirect Nanoparticles as synthetic vaccines Josiah D Smith1, Logan D Morton1 and Bret D Ulery As vaccines have transitioned from the use of whole pathogens to only the required antigenic epitopes, unwanted side effects have been decreased, but corresponding immune responses have been greatly diminished. To enhance immunogenicity, a variety of controlled release vehicles have been proposed as synthetic vaccines, but nanoparticles have emerged as particularly impressive systems due to many exciting publications. In specific, nanoparticles have been shown capable of not only desirable vaccine release, but can also be targeted to immune cells of interest, loaded with immunostimulatory substances termed adjuvants, or even induce desirable immune activating effects on their own. In the present review, recent advances in the utilization of inorganic, polymeric, and biomolecular nanoparticles as synthetic vaccines are discussed. Address Department of Chemical Engineering, University of Missouri, Columbia, MO 65211, United States Corresponding author: Ulery, Bret D ([email protected]) J.D.S. and L.D.M. contributed equally to this work.
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Current Opinion in Biotechnology 2015, 34:217–224 This review comes from a themed issue on Nanobiotechnology Edited by Igor L Medintz and Matthew Tirrell
http://dx.doi.org/10.1016/j.copbio.2015.03.014 0958-1669/# 2015 Elsevier Ltd. All rights reserved.
Introduction Over the past 200 years, the advancement of vaccines is one of humankind’s greatest achievements leading to the eradication of small pox worldwide and polio from the developed world as well as the near elimination of other deadly infectious diseases like diphtheria. Traditionally, vaccines have been comprised of whole-killed or live, attenuated pathogens which provide not only the necessary protective antigens, but also a wide variety of additional pathogenic components which enhance the host immune response. While successful, these vaccines provide many unnecessary components (i.e. lipopolysaccharides) that can induce undesirable or even toxic side effects. Recent research has focused on using only the required protein and peptide antigens which has greatly decreased vaccineassociated complications but also lessened corresponding immune responses since the antigens alone are weak www.sciencedirect.com
immunogens due to their fragile nature and inability to provide secondary immunostimulatory signals termed adjuvants. One emerging methodology to address both of these drawbacks is the utilization of nanoparticles (NPs) which can achieve directed and/or controlled release of associated antigens while either co-delivering known adjuvants or acting as adjuvants themselves. This review will focus on the potential of NPs to act as synthetic vaccines to enhance corresponding immune responses highlighting three major categories of NPs: inorganic, polymeric, and biomolecular (Figure 1).
Inorganic nanoparticles The use of inorganic NPs as vaccines is a developing field in immunology as evidenced by a variety of significant recent publications. Inorganic NPs have been used as both adjuvants of and delivery vehicles for antigens in order to enhance the immune response. Structurally, inorganic NPs used in vaccines consist of a solid nanoparticle core that can be conjugated with antigens. Four categories of inorganic NPs will be discussed, specifically highlighting their applications in vitro and in vivo: aluminum-based (AlNPs), gold (AuNPs), silicate (SiNPs), and calcium phosphate (CaPNPs) nanoparticles.
Aluminum-based AlNP adjuvants are commonly used in vaccines to facilitate strong antigen-specific immune responses. For example, tetanus, diphtheria, and influenza type b vaccines all make use of the adjuvanticity of AlNPs [1]. Beyond innate immunostimulatory effects, aluminum salts have been shown to promote an immune response to substances that would not usually initiate such a response [2]. A study regarding the effect of different aluminum oxyhydroxide nanoparticles provides evidence that the most effective shape for this aluminum adjuvant is rodlike. In specific, rod-like AlNPs stimulated a stronger dendritic cell response than spherical AlNPs in vitro which correlated to a more robust immune response against co-delivered antigen in vivo [3].
Gold AuNPs hold particular promise in vaccinology due to their biocompatibility and highly modifiable surface. As an example of their facile surface chemistry, simple oxidation of AuNPs by sulfur groups found in protein epitopes makes antigen conjugation and the development of novel nanoparticle vaccines very straightforward [4]. Beyond their use as a peptide delivery platform, AuNPs have also been shown to increase the effectiveness of non-traditional exposure to antigens. Specifically, chitosan modified AuNPs loaded with tetanus toxoid administered Current Opinion in Biotechnology 2015, 34:217–224
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orally were substantially more effective at stimulating an immune response than tetanus toxoid alone [5]. Due to the stability of AuNPs, a scaffold of complex surface chemistries can be added stepwise, as used by Gregory et al. in developing complex peptide-saccharide conjugated AuNPs able to convey immunological protection against glanders [6].
Silicate SiNPs have been recently used as antigen carriers to enable controlled antigen release in vivo. This technique has been shown to increase the magnitude of both the humoral and cell-mediated immune response in mice against porcine circovirus type 2 [7]. An interesting study regarding SiNPs demonstrated that the antigenic conjugation of these particles is not required to convey their immunostimulatory effect [8].
significant surface modification [9]. CaPNPs can be used as a central binding site for antigenic proteins. Zhou et al. used an inorganic core surrounded by a large protein containing a CaP-binding region to effectively stimulate a short-term immune response to influenza [10]. Due to the simple, versatile chemistry of these nanoparticles, complex multilayered nanoparticles embedded with antigenic epitopes and adjuvants have been synthesized, leading to enhanced short-term and long-term immune responses when compared to traditional nanoparticle-independent antigenic vaccines as shown in Figure 2 [11]. While inorganic nanoparticles have been shown to adjuvant immune response effectively, they are limited in chemistry and physical properties. Polymeric nanoparticles, with widely varied chemistries, have been tested and used as adjuvants and vaccine delivery systems.
Calcium phosphate CaPNPs are promising candidates for vaccine applications due to several of their characteristics: they can decompose in vivo into components that are non-toxic and bioresorbable, and they display facile surface chemistry allowing for
Polymeric nanoparticles Polymeric NPs are of immense interest for vaccinology due to their biocompatibility, predictable degradation, and diverse chemistry. Due to the wide variety of polymeric
Figure 1
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Nanoparticle-based synthetic vaccines. Inorganic nanoparticles (a), polymeric nanoparticles (b), and biomolecular nanoparticles including liposomes (c), virus-like particles (d), micelles (e), and immunostimulatory complexes (f) represent a wide range of nanomaterials that have been utilized as synthetic vaccine systems.Adapted from [26]. Current Opinion in Biotechnology 2015, 34:217–224
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Figure 2
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Calcium phosphate nanoparticle adjuvanticity. Multilayered calcium phosphate nanoparticle were conjugated with both protein antigens and adjuvants (a). Scanning electron microscopy revealed multiple layer calcium phosphate deposition and particle conjugation yields small angular nanoparticles less than 50 nm in diameter (b). Conjugating CpG adjuvant and gp70 and GagL antigens to CaP yielded significantly greater numbers of active CD4+ and CD8+ T cells as determined by ELISPOT assay (c). Adapted from [11] with the permission of Elsevier.
NPs under study, this review will focus on several promising candidates for vaccine applications generalized into the classes of naturally derived polymers, polyesters, and polyanhydrides.
One interesting use of chitosan nanoparticles is to enhance mucosal vaccination as an alternative to classical intramuscular or subcutaneous routes which has been shown effective in fish against the virus Vibrio parahaemolyticus [13].
Naturally derived polymers
Chitosan, or poly-D-glucosamine, is a naturally-derived biodegradable polymer that has shown great ability to stimulate an adaptive immune response. Chitosan nanoparticles have been used in conjunction with a DNA adjuvant to improve an antigen-specific immunity [12]. www.sciencedirect.com
Hyaluronic acid (HA) is a promising candidate for vaccine nanoparticles as it is biodegradable, native to the human body, and is being investigated in cancer therapy clinical trials [14]. HA is composed of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine, with Current Opinion in Biotechnology 2015, 34:217–224
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its native biological role largely dependent on the length of the polysaccharide [15]. By using trimethyl chitosan in conjunction with HA to form a more physiologically stable nanoparticle to house antigen, the IgG antibody titer increased compared to both non-conjugated nanoparticles and free antigen [16]. Polyesters
Poly(glycolic acid) (PGA) is a synthetically created polymer comprised of repeating glycolide monomers that degrades into biocompatible glycolic acid. It has been used as an alternative to more harmful adjuvants such as aluminum salts to lengthen in vivo antigenic exposure time [17]. While promising, PGA is often highly crystalline and degrades quite slowly limiting its utility as a vaccine delivery system. Poly(lactic-co-glycolic acid) (PLGA) is a synthetically produced polyester similar to PGA comprised of lactide and glycolide monomers that degrades into biocompatible lactic acid and glycolic acid. Due to the random sequence of the monomers, PLGA is much less crystalline yielding quicker degradation times that PGA or poly(lactic acid) (PLA). PLGA nanoparticles are wellstudied and have been shown to stimulate an immune response [18]. A novel application of these immunogenic nanoparticles is in intranasal vaccination of pigs, where they increased both humoral and cell-mediated immune responses [19]. These nanoparticles have been used in conjugation with a lipid monolayer to facilitate slower antigen release in vitro, a promising method combining the ease of lipid nanoparticle synthesis with the diversity of polymeric nanoparticle chemistry [20].
Polyanhydrides Polyanhydrides are degradable synthetic polymers that possess highly hydrolytically sensitive anhydride bonds which allows for a much greater diversity of chemistry backbones to be chosen for drug delivery applications when compared to polyesters. CPH:SA and CPTEG:CPH are two polyanhydride copolymer systems used for vaccine applications today. They are promising candidates as nanoparticulate vaccine carriers due to their simple and reliable erosion kinetics, tunable payload release, and capacity to maintain viable protein delivery in vivo [21]. Pneumococcal surface protein A was encapsulated and delivered to mice using 20:80 CPH:SA and 50:50 CPTEG:CPH nanoparticles which induced a strong antibody response against the antigen [22]. Nanoparticles fabricated from 50:50 CPTEG:CPH were also used to deliver aGal-modified antigen which slowly diffused from the degrading nanoparticle leading to a strong CD4+ T cell response [23]. The mechanism responsible for polyanhydride nanoparticle adjuvanticity has in part been ascribed to their uptake by dendritic cells, but specific antigenic processing pathways have yet to be elucidated [24]. Current Opinion in Biotechnology 2015, 34:217–224
Beyond the polymeric NPs covered in this review, there are many more currently being designed and tested for vaccine applications. For polymeric NPs that have been published in the literature, two areas that still need further investigation are in vivo processing mechanisms (e.g. innate immune cell uptake, trafficking of NPs to draining lymph nodes) and a comparison of the effectiveness of different polymeric NPs.
Biomolecular nanoparticles While both polymeric and inorganic materials have been successfully utilized for nanoparticle-based vaccine delivery, biomolecular materials possess many positive attributes which make them exciting candidates for use in synthetic vaccine applications. Biomolecular materials are defined as biologically-inspired systems that utilize biomolecules as their foundation. A significant advantage of biomolecular materials is that they can be tailored to deliver a variety of payloads and display different moieties on their surfaces which provides them with significant potential as synthetic vaccines [25]. While a variety of biomolecular-based nanoparticles have been studied, the four most commonly used for vaccine applications are liposomes, virus-like particles (VLPs), micelles, and immunostimulating complexes (ISCOMs) [26].
Liposomes Liposomes are spherical nanoparticles comprised of a lamellar lipid bilayer or multiple lipid bilayers that mimic vesicles found within cells [27]. Lipids are biomolecules that possess a hydrophilic head and a hydrophobic tail or tails which under the correct conditions self-assemble in water to form liposomes possessing hydrophilic inner and outer membranes. The liposome core is hydrophilic whereas the bilayer interior is hydrophobic which allows for liposomes to encapsulate a variety of drugs and be utilized for controlled payload delivery applications. Liposomes have shown significant clinical potential since they are biocompatible, stable within the body, and can be modified to display targeting moieties [28]. Significant research has been published in the literature demonstrating the vaccination potential of liposomes. A particular advantage of liposomes is that they can be modified to achieve desirable immunostimulatory properties. By modulating liposomes to present lectin binding mannose on their surface as well as entrap monophosphoryl lipid A (MPLA) adjuvant, a novel nanovaccine system was developed that was capable of targeting dendritic cells and facilitating enhanced antigen presentation to T cells against a model antigen [29]. While liposomes have been utilized as vaccine delivery vehicles against a number of pathogens, one particularly interesting application is in prophylaxis against tuberculosis (TB), a debilitating and deadly infectious disease that infects approximately one-third of the world population. MPLA-modified liposomes loaded with a fusion protein www.sciencedirect.com
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comprised of multiple TB epitopes induced strong T cellmediated immune responses in mice [30]. This is a particularly exciting result since previous TB vaccines have facilitated strong B cell-mediated immune responses which have been found minimally efficacious at preventing subsequent TB infections. A next generation liposome technology that has been utilized for vaccination is the virosome. Virosomes are liposomes that display viral proteins (e.g. influenza-derived hemagglutinin and neuraminidase) on their surface which allow for them to actively fuse with target cells [31]. These complex drug delivery systems possess the versatility of liposomes with highly specific targeting capabilities. Virosomes have been utilized clinically for a variety of prophylactic applications including as tetanus and hepatitis B vaccines [31]. Recently, it has been discovered that virosomal vaccines can be designed to preferentially activate cytotoxic T cells to improve vaccine-based protection against influenza-infected host cells [32].
Virus-like particles Similar to virosomes are a class of biomolecular nanoparticles termed virus-like-particles (VLPs). The inherent difference between the two is that virosomes possess a small concentration of certain viral proteins presented on the surface of liposomes whereas VLPs resemble the traditional structure of viruses (i.e. complete envelope or capsid) except they do not possess any viral genetic material making them non-infectious and incapable of replicating. VLPs can be formed from either one or a combination of viral proteins which can be rapidly engineered to counter pathogenic outbreaks [33,34]. VLPs work well as synthetic vaccines because they possess an external viral shell which the body immediately recognizes as foreign. Quick and effective processing of VLPs yields a rapid and durable host immune response [35]. The first VLP-based human vaccine conveys protection against hepatitis B and has been widely administered since its release in 1986 [36]. More recently VLPs have been utilized as delivery systems for multiple Human Papillomavirus vaccines (HPV) including those marketed as Gardasil and Cervarix. Recent research has shown that VLP-based HPV vaccines induce a highly cross-protective antibody response that conveys protection against a vast majority of HPV serotypes making VLPs robustly effective [37]. Cytotoxic T cells play a key role in clearing infected cells and controlling pathogen load during chronic infection. By targeting this T cell response, VLPs can be very effectively utilized in vaccines.
Micelles While liposomes and VLPs have hydrophilic cores, another group of biomolecular nanoparticles termed micelles possess a hydrophobic core. Micelles form when amphiphilic molecules are solubilized and self-assemble www.sciencedirect.com
by shielding their hydrophobic moieties in their core and displaying the hydrophilic moieties on their surface [38]. Micelles are commonly utilized to deliver poorly water soluble drugs encapsulated in their cores or entrapped amphiphilic molecules [39]. Micelles have been utilized as vaccine delivery systems via two common methods. First, protein vaccines can be easily tethered covalently to the hydrophilic micelle corona. This method has been utilized to attach HIV vaccines to adjuvant-loaded polymer-based micelles yielding strong antigen presenting cell activation in vitro [40]. A similar strategy has been employed for model peptide antigen attached to adjuvant-loaded polymerlipid micelles which were shown to preferentially bind albumin and traffic to the lymph node where the greatly enhanced T cell priming and functioned as an anti-cancer vaccine [41]. Alternatively, peptide amphiphiles can be synthesized by covalently attaching hydrophilic peptide vaccines to hydrophobic moieties (i.e. lipids or aliphatic hydrocarbons) which will self-assemble into micelles in water. By utilizing the vaccine as part of the nanoparticle, peptide amphiphile micelles can achieve antigenic payloads of up to 90% by weight [42]. Peptide amphiphile micelles have been found to induce strong anti-tumor cytotoxic T cell responses and antibody responses against Streptococcus pyogenes in mice without the need for adjuvant supplementation [43,44]. Micelle modulatory provides a unique platform to enhance their capacity as synthetics vaccines. By choosing unique hydrophobic core chemistries, polymer-based micelles can be used to trigger endosome disruption which allows for vaccines to be delivered to the cytosol of cells which yielded antigenic cross-presentation and strong cytotoxic T cell responses in vivo [45]. Also, different amphiphiles with the same or similar hydrophobic moieties are able to self-assemble into heterogeneous micelles. This effect has been leveraged to create peptide amphiphile micelles capable of delivering multiple antigens to the same cell as demonstrated for vaccines against the Herpes simplex virus [46]. Immunostimulating complexes
Similar to micelles, immunostimulating complexes (ISCOMs) are self-assembled structures comprised of biomolecular components (i.e. cholesterols, phospholipids, and Quil A saponins), but self-assembly of the components facilitates the formation of cage-like structures that can be used to entrap adjuvants instead of the hydrophobic core found in micelles. The immunostimulatory function of ISCOMs is due to the fact that many of the heterogeneous mixtures possess Quil A saponins which are known potent adjuvants. While ISCOMs have been studied for nearly 30 years and have been shown quite potent as synthetic vaccine [47], they can induce substantial, undesirable injection-site reactions [48] Current Opinion in Biotechnology 2015, 34:217–224
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which have relegated their development almost exclusively to animal vaccines [49].
4.
A new iteration of ISCOMs termed ISCOMATRIX is similar to the classical ISCOM except the Quil A saponins are first purified to yield a defined group of saponins that do not induce significant inflammation nor toxic side effects [50]. Recent research has shown that ISCOMATRIX can induce both antibody-mediated and cytotoxicmediated immune responses [51] as well as induce HIVspecific immune responses in mice and rabbits [52].
5.
Barhate G, Gautam M, Gairola S, Jadhav S, Pokharkar V: Enhanced mucosal immune responses against tetanus toxoid using novel delivery system comprised of chitosanfunctionalized gold nanoparticles and botanical adjuvant: characterization, immunogenicity, and stability assessment. J Pharm Sci 2014, 11:3448-3456. This in vitro study highlights the combination of two traditionally used biomaterials — chitosan and gold, which when formed into a composite nanparticle greatly enhanced the immune response. 6.
Gregory AE, Judy BM, Qazi O, Blumentritt CA, Brown KA, Shaw AM, Torres AG, Titball RW: A gold nanoparticle-linked glycoconjugate vaccine against Burkholderia mallei. Nanomedicine 2015, 11:447-456.
7.
Guo HC, Feng XM, Sun SQ, Wei YQ, Sun DH, Liu XT, Liu ZX, Luo JX, Yin H: Immunization of mice by hollow mesoporous silica nanoparticles as carriers of porcine circovirus type 2 ORF2 protein. Virol J 2012, 9:108.
8.
Wibowo N, Chuan YP, Seth A, Cordoba Y, Lua LH, Middelberg AP: Co-administration of non-carrier nanoparticles boosts antigen immune response without requiring protein conjugation. Vaccine 2014, 32:3664-3669.
9.
Temchura VV, Kozlova D, Sokolova V, Uberla K, Epple M: Targeting and activation of antigen-specific B-cells by calcium phosphate nanoparticles loaded with protein antigen. Biomaterials 2014, 35:6098-6105.
Conclusions and outlook Over the past decade, significant advancements in synthetic organic chemistry, device fabrication, and molecular-based targeting have let to the generation of a wide range of NPs that can be utilized for a variety of biomedical applications. No field is this more apparent than in vaccinology where inorganic, polymeric, and biomolecular NPs have all been shown to improve weakly immunogenic antigens like peptides and proteins. NP-based synthetic vaccines for prophylaxis against pathogenic infections are currently being motivated into the clinic. While impressive, new applications of NPs as therapeutic and tolerogenic vaccines in the treatment of cancer and autoimmune diseases, respectively, provide even greater possibility for this synthetic vaccine platform. While some generalized fears regarding the implications and toxicity of NPs still remain in the general population, research described in this review supplemented by an archive of studies demonstrating NP biocompatibility and utility in biomedical applications is changing the public perception for the better which will allow for wide adoption of NP-based medical technologies.
Acknowledgements LM thanks the Discovery Fellows Program at the University of Missouri for their support. BDU gratefully acknowledges support from start-up funds from the University of Missouri.
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21. Ulery BD, Petersen LK, Phanse Y, Kong CS, Broderick SR, Kumar D, Ramer-Tait AE, Carrillo-Conde B, Rajan K, Wannemuehler MJ et al.: Rational design of pathogenmimicking amphiphilic materials as nanoadjuvants. Sci Rep 2011, 1:198. 22. Haughney SL, Petersen LK, Schoofs AD, Ramer-Tait AE, King JD, Briles DE, Wannemuehler MJ, Narasimhan B: Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles. Acta Biomater 2013, 9:8262-8271. 23. Phanse Y, Carrillo-Conde BR, Ramer-Tait AE, Broderick S, Kong CS, Rajan K, Flick R, Mandell RB, Narasimhan B, Wannemuehler MJ: A systems approach to designing next generation vaccines: combining alpha-galactose modified antigens with nanoparticle platforms. Sci Rep 2014, 4:3775. While many research articles focus on one very specific nanoparticle formulation, this article takes a more complete approach by testing several modification to their nanoparticle synthetic vaccine to identify the most effective candidates. 24. Torres MP, Wilson-Welder JH, Lopac SK, Phanse Y, CarrilloConde B, Ramer-Tait AE, Bellaire BH, Wannemuehler MJ, Narasimhan B: Polyanhydride microparticles enhance dendritic cell antigen presentation and activation. Acta Biomater 2011, 7:2857-2864. 25. Zhao L, Wibowo SA, Zhao N, Mitter CX, Yu N, Middleburg CAPJ: Nanoparticle vaccines. Vaccine 2014, 32:327-337. This paper gives a brief overview of some of the ways that nanoparticles are being used or could be used in the future in vaccination applications. 26. Gregory AE, Williamson TRD: Vaccine delivery using nanoparticles. Cell Infect Microbiol 2013, 3:1-13. 27. Kraft JC, Wang FJ, Ho ZRJY: Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. J Pharm Sci 2014, 103:29-52. 28. RAS: Liposomes as vaccine delivery systems: a review of the recent advances. Therap Adv Vaccines 2014, 2:159-182. 29. Wang N, Zhang WT, Chen M, Niu R, Deng RY: Mannose derivative and lipid A dually decorated cationic liposomes as an effective cold chain free oral mucosal vaccine adjuvant-delivery system. Eur J Pharm Biopharm 2014, 88:194-206. 30. Orr MT, Baldwin FC, Sivananthan SL, Lucas SJ, Lin E, Phan S, Moon T, Vedvick JJ, Reed TS, Coler SGRN: Adjuvant formulation structure and composition are critical for the development of an effective vaccine against tuberculosis. J Control Release 2013, 172:190-200. This research demonstrates the capacity of how liposomes can be used in vaccination specifically highlighting their potential in the prophylactic protection against tuberculosis. 31. Felnerova D, Gluck VJ, Moser RC: Liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs. Curr Opin Biotechnol 2004, 15:518-529. 32. Monto AS, Aspinall AF, McElhaney R, Montan˜o JE, Nichol LF, Puig-Barbara` KL, Schmitt J, Stephenson JI: Influenza control in the 21st century: optimizing protection of older adults. Vaccine 2009, 27:5043-5053. 33. Srinivasan A, Ayyavoo RA, Srivastava VS: Nanotechnologybased approaches for the development of diagnostics, www.sciencedirect.com
36. Grimm SKAM: Vaccine design: emerging concepts and renewed optimism. Curr Opin Biotechnol 2013, 24:1078-1088. 37. Bissett SL, Mattiuzzo G, Draper E, Godi A, Wilkinson DE, Minor P, Page M, Beddows S: Pre-clinical immunogenicity of human papillomavirus alpha-7 and alpha-9 major capsid proteins. Vaccine 2014, 33:6548-6555. The results presented in this paper show exciting potential for virus-like particles to be used in vaccine applications like preventing the spread of human papillomavirus. 38. Zhang N, Bader WPRA: Polysaccharide-based micelles for drug delivery. Pharmaceutics 2013, 5:329-352. 39. VPT: Structure and design of polymeric surfactant-based drug delivery systems. J Control Release 2001, 73:137-172. 40. Jime´nez-Sa´nchez G, Chane-Haong PV, Handke´ C, Terrat N, Gigmes C, Trimaille D, Verrier TB: Preparation and in vitro evaluation of imiquimod loaded polylactide-based micelles as potential vaccine adjuvants. Pharm Res 2014, 32:311-320. 41. Liu H, Zheng MK, Szeto Y, Li GL, Huang AV, Van Egeren B, Park DS, Irvine CDJ: Structure-based programming of lymph-node targeting in molecular vaccines. Nature 2014, 507:519-521. 42. Black M, Olive TA, Tirrell CM: Advances in the design and delivery of peptide subunit vaccines with a focus on Toll-like receptor agonists. Exp Rev Vaccines 2010, 9:157-173. 43. Black M, Trent A, Kostenko Y, Lee JS, Olive C, Tirrell M: Self assembled peptide amphiphile micelles containing a cytotoxic T-cell epitope promote a protective immune response in vivo. Adv Mater 2012, 24:3845-3849. This in vivo research focused on using micelles to induce anti-cancer cytotoxic T cells responses. This paper was particularly interesting since it shows how micelles are more effective than traditional adjuvanted formulations and could revolutionize vaccination through a nanoparticle platform. 44. Trent A, Ulery BD, Black MJ, Barrett JC, Liang S, Kostenko Y, David NA, Tirrell MV: Peptide amphiphile micelles selfadjuvant group A streptococcal vaccination. AAPS J 2014, 17:380-388. 45. Keller S, Patilea WJ, Kern GI, Convertine HB, Stayton AJPS: Neutral polymer micelle carriers with pH-responsive, endosome-releasing activity modulate antigen trafficking to enhance CD8+ T cell responses. J Control Release 2014, 191:24-33. 46. Accardo A, Tesauro VM, Galdiero D, Finamore M, Martora E, Mansi F, Ringhieri R, Morelli PG: Self-assembled or mixed peptide amphiphile micelles from Herpes simplex virus glycoproteins as potential immunomodulatory treatment. Int J Nanomed 2014, 9:2137-2148. 47. Sanders MT, Deliyannis BL, Pearse GMJ: ISCOMTM-based vaccines: the second decade. Immunol Cell Biol 2005, 83:119-128. 48. Waite DC, Ennis JE, Edelman FA, White R, Kammer B, Anderson R, Kensil CCR: Three double-blind, randomized trials evaluating the safety and tolerance of different formulations of the saponin adjuvant QS-21. Vaccine 2001, 19:3957-3967. 49. Ha¨gglund S, Blodo¨rn HK, Makabi-Panzu K, Gaillard B, Ellencrona AL, Chevret K, Hellman D, Bengtsson L, Riffault KL, Taylor S et al.: Characterization of an experimental vaccine for bovine respiratory syncytial virus. Clin Vaccine Immunol 2014, 21:997-1004. Current Opinion in Biotechnology 2015, 34:217–224
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This paper shows ISCOMs can be employed to induce a strong and durable immune response against bovine respiratory syncytial virus.
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therapeutic vaccines against infectious diseases. J Med Microbiol 2012, 61:935-943. 52. Buglione-Corbett R, Marty-Roix PK, Li R, West W, Wang K, Morelli S, Lien AB, Lu ES: Reduced MyD88 dependency of ISCOMATRIX TM adjuvant in a DNA prime-protein boost HIV vaccine. Hum Vaccines Immunotherap 2014, 10:1078-1090.
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ScienceDirect Nanoparticles as synthetic vaccines Josiah D Smith1, Logan D Morton1 and Bret D Ulery As vaccines have transitioned from the use of whole pathogens to only the required antigenic epitopes, unwanted side effects have been decreased, but corresponding immune responses have been greatly diminished. To enhance immunogenicity, a variety of controlled release vehicles have been proposed as synthetic vaccines, but nanoparticles have emerged as particularly impressive systems due to many exciting publications. In specific, nanoparticles have been shown capable of not only desirable vaccine release, but can also be targeted to immune cells of interest, loaded with immunostimulatory substances termed adjuvants, or even induce desirable immune activating effects on their own. In the present review, recent advances in the utilization of inorganic, polymeric, and biomolecular nanoparticles as synthetic vaccines are discussed. Address Department of Chemical Engineering, University of Missouri, Columbia, MO 65211, United States Corresponding author: Ulery, Bret D ([email protected]) J.D.S. and L.D.M. contributed equally to this work.
1
Current Opinion in Biotechnology 2015, 34:217–224 This review comes from a themed issue on Nanobiotechnology Edited by Igor L Medintz and Matthew Tirrell
http://dx.doi.org/10.1016/j.copbio.2015.03.014 0958-1669/# 2015 Elsevier Ltd. All rights reserved.
Introduction Over the past 200 years, the advancement of vaccines is one of humankind’s greatest achievements leading to the eradication of small pox worldwide and polio from the developed world as well as the near elimination of other deadly infectious diseases like diphtheria. Traditionally, vaccines have been comprised of whole-killed or live, attenuated pathogens which provide not only the necessary protective antigens, but also a wide variety of additional pathogenic components which enhance the host immune response. While successful, these vaccines provide many unnecessary components (i.e. lipopolysaccharides) that can induce undesirable or even toxic side effects. Recent research has focused on using only the required protein and peptide antigens which has greatly decreased vaccineassociated complications but also lessened corresponding immune responses since the antigens alone are weak www.sciencedirect.com
immunogens due to their fragile nature and inability to provide secondary immunostimulatory signals termed adjuvants. One emerging methodology to address both of these drawbacks is the utilization of nanoparticles (NPs) which can achieve directed and/or controlled release of associated antigens while either co-delivering known adjuvants or acting as adjuvants themselves. This review will focus on the potential of NPs to act as synthetic vaccines to enhance corresponding immune responses highlighting three major categories of NPs: inorganic, polymeric, and biomolecular (Figure 1).
Inorganic nanoparticles The use of inorganic NPs as vaccines is a developing field in immunology as evidenced by a variety of significant recent publications. Inorganic NPs have been used as both adjuvants of and delivery vehicles for antigens in order to enhance the immune response. Structurally, inorganic NPs used in vaccines consist of a solid nanoparticle core that can be conjugated with antigens. Four categories of inorganic NPs will be discussed, specifically highlighting their applications in vitro and in vivo: aluminum-based (AlNPs), gold (AuNPs), silicate (SiNPs), and calcium phosphate (CaPNPs) nanoparticles.
Aluminum-based AlNP adjuvants are commonly used in vaccines to facilitate strong antigen-specific immune responses. For example, tetanus, diphtheria, and influenza type b vaccines all make use of the adjuvanticity of AlNPs [1]. Beyond innate immunostimulatory effects, aluminum salts have been shown to promote an immune response to substances that would not usually initiate such a response [2]. A study regarding the effect of different aluminum oxyhydroxide nanoparticles provides evidence that the most effective shape for this aluminum adjuvant is rodlike. In specific, rod-like AlNPs stimulated a stronger dendritic cell response than spherical AlNPs in vitro which correlated to a more robust immune response against co-delivered antigen in vivo [3].
Gold AuNPs hold particular promise in vaccinology due to their biocompatibility and highly modifiable surface. As an example of their facile surface chemistry, simple oxidation of AuNPs by sulfur groups found in protein epitopes makes antigen conjugation and the development of novel nanoparticle vaccines very straightforward [4]. Beyond their use as a peptide delivery platform, AuNPs have also been shown to increase the effectiveness of non-traditional exposure to antigens. Specifically, chitosan modified AuNPs loaded with tetanus toxoid administered Current Opinion in Biotechnology 2015, 34:217–224
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orally were substantially more effective at stimulating an immune response than tetanus toxoid alone [5]. Due to the stability of AuNPs, a scaffold of complex surface chemistries can be added stepwise, as used by Gregory et al. in developing complex peptide-saccharide conjugated AuNPs able to convey immunological protection against glanders [6].
Silicate SiNPs have been recently used as antigen carriers to enable controlled antigen release in vivo. This technique has been shown to increase the magnitude of both the humoral and cell-mediated immune response in mice against porcine circovirus type 2 [7]. An interesting study regarding SiNPs demonstrated that the antigenic conjugation of these particles is not required to convey their immunostimulatory effect [8].
significant surface modification [9]. CaPNPs can be used as a central binding site for antigenic proteins. Zhou et al. used an inorganic core surrounded by a large protein containing a CaP-binding region to effectively stimulate a short-term immune response to influenza [10]. Due to the simple, versatile chemistry of these nanoparticles, complex multilayered nanoparticles embedded with antigenic epitopes and adjuvants have been synthesized, leading to enhanced short-term and long-term immune responses when compared to traditional nanoparticle-independent antigenic vaccines as shown in Figure 2 [11]. While inorganic nanoparticles have been shown to adjuvant immune response effectively, they are limited in chemistry and physical properties. Polymeric nanoparticles, with widely varied chemistries, have been tested and used as adjuvants and vaccine delivery systems.
Calcium phosphate CaPNPs are promising candidates for vaccine applications due to several of their characteristics: they can decompose in vivo into components that are non-toxic and bioresorbable, and they display facile surface chemistry allowing for
Polymeric nanoparticles Polymeric NPs are of immense interest for vaccinology due to their biocompatibility, predictable degradation, and diverse chemistry. Due to the wide variety of polymeric
Figure 1
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Nanoparticle-based synthetic vaccines. Inorganic nanoparticles (a), polymeric nanoparticles (b), and biomolecular nanoparticles including liposomes (c), virus-like particles (d), micelles (e), and immunostimulatory complexes (f) represent a wide range of nanomaterials that have been utilized as synthetic vaccine systems.Adapted from [26]. Current Opinion in Biotechnology 2015, 34:217–224
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Figure 2
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+ CpG GagL/gp70
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Calcium phosphate nanoparticle adjuvanticity. Multilayered calcium phosphate nanoparticle were conjugated with both protein antigens and adjuvants (a). Scanning electron microscopy revealed multiple layer calcium phosphate deposition and particle conjugation yields small angular nanoparticles less than 50 nm in diameter (b). Conjugating CpG adjuvant and gp70 and GagL antigens to CaP yielded significantly greater numbers of active CD4+ and CD8+ T cells as determined by ELISPOT assay (c). Adapted from [11] with the permission of Elsevier.
NPs under study, this review will focus on several promising candidates for vaccine applications generalized into the classes of naturally derived polymers, polyesters, and polyanhydrides.
One interesting use of chitosan nanoparticles is to enhance mucosal vaccination as an alternative to classical intramuscular or subcutaneous routes which has been shown effective in fish against the virus Vibrio parahaemolyticus [13].
Naturally derived polymers
Chitosan, or poly-D-glucosamine, is a naturally-derived biodegradable polymer that has shown great ability to stimulate an adaptive immune response. Chitosan nanoparticles have been used in conjunction with a DNA adjuvant to improve an antigen-specific immunity [12]. www.sciencedirect.com
Hyaluronic acid (HA) is a promising candidate for vaccine nanoparticles as it is biodegradable, native to the human body, and is being investigated in cancer therapy clinical trials [14]. HA is composed of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine, with Current Opinion in Biotechnology 2015, 34:217–224
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its native biological role largely dependent on the length of the polysaccharide [15]. By using trimethyl chitosan in conjunction with HA to form a more physiologically stable nanoparticle to house antigen, the IgG antibody titer increased compared to both non-conjugated nanoparticles and free antigen [16]. Polyesters
Poly(glycolic acid) (PGA) is a synthetically created polymer comprised of repeating glycolide monomers that degrades into biocompatible glycolic acid. It has been used as an alternative to more harmful adjuvants such as aluminum salts to lengthen in vivo antigenic exposure time [17]. While promising, PGA is often highly crystalline and degrades quite slowly limiting its utility as a vaccine delivery system. Poly(lactic-co-glycolic acid) (PLGA) is a synthetically produced polyester similar to PGA comprised of lactide and glycolide monomers that degrades into biocompatible lactic acid and glycolic acid. Due to the random sequence of the monomers, PLGA is much less crystalline yielding quicker degradation times that PGA or poly(lactic acid) (PLA). PLGA nanoparticles are wellstudied and have been shown to stimulate an immune response [18]. A novel application of these immunogenic nanoparticles is in intranasal vaccination of pigs, where they increased both humoral and cell-mediated immune responses [19]. These nanoparticles have been used in conjugation with a lipid monolayer to facilitate slower antigen release in vitro, a promising method combining the ease of lipid nanoparticle synthesis with the diversity of polymeric nanoparticle chemistry [20].
Polyanhydrides Polyanhydrides are degradable synthetic polymers that possess highly hydrolytically sensitive anhydride bonds which allows for a much greater diversity of chemistry backbones to be chosen for drug delivery applications when compared to polyesters. CPH:SA and CPTEG:CPH are two polyanhydride copolymer systems used for vaccine applications today. They are promising candidates as nanoparticulate vaccine carriers due to their simple and reliable erosion kinetics, tunable payload release, and capacity to maintain viable protein delivery in vivo [21]. Pneumococcal surface protein A was encapsulated and delivered to mice using 20:80 CPH:SA and 50:50 CPTEG:CPH nanoparticles which induced a strong antibody response against the antigen [22]. Nanoparticles fabricated from 50:50 CPTEG:CPH were also used to deliver aGal-modified antigen which slowly diffused from the degrading nanoparticle leading to a strong CD4+ T cell response [23]. The mechanism responsible for polyanhydride nanoparticle adjuvanticity has in part been ascribed to their uptake by dendritic cells, but specific antigenic processing pathways have yet to be elucidated [24]. Current Opinion in Biotechnology 2015, 34:217–224
Beyond the polymeric NPs covered in this review, there are many more currently being designed and tested for vaccine applications. For polymeric NPs that have been published in the literature, two areas that still need further investigation are in vivo processing mechanisms (e.g. innate immune cell uptake, trafficking of NPs to draining lymph nodes) and a comparison of the effectiveness of different polymeric NPs.
Biomolecular nanoparticles While both polymeric and inorganic materials have been successfully utilized for nanoparticle-based vaccine delivery, biomolecular materials possess many positive attributes which make them exciting candidates for use in synthetic vaccine applications. Biomolecular materials are defined as biologically-inspired systems that utilize biomolecules as their foundation. A significant advantage of biomolecular materials is that they can be tailored to deliver a variety of payloads and display different moieties on their surfaces which provides them with significant potential as synthetic vaccines [25]. While a variety of biomolecular-based nanoparticles have been studied, the four most commonly used for vaccine applications are liposomes, virus-like particles (VLPs), micelles, and immunostimulating complexes (ISCOMs) [26].
Liposomes Liposomes are spherical nanoparticles comprised of a lamellar lipid bilayer or multiple lipid bilayers that mimic vesicles found within cells [27]. Lipids are biomolecules that possess a hydrophilic head and a hydrophobic tail or tails which under the correct conditions self-assemble in water to form liposomes possessing hydrophilic inner and outer membranes. The liposome core is hydrophilic whereas the bilayer interior is hydrophobic which allows for liposomes to encapsulate a variety of drugs and be utilized for controlled payload delivery applications. Liposomes have shown significant clinical potential since they are biocompatible, stable within the body, and can be modified to display targeting moieties [28]. Significant research has been published in the literature demonstrating the vaccination potential of liposomes. A particular advantage of liposomes is that they can be modified to achieve desirable immunostimulatory properties. By modulating liposomes to present lectin binding mannose on their surface as well as entrap monophosphoryl lipid A (MPLA) adjuvant, a novel nanovaccine system was developed that was capable of targeting dendritic cells and facilitating enhanced antigen presentation to T cells against a model antigen [29]. While liposomes have been utilized as vaccine delivery vehicles against a number of pathogens, one particularly interesting application is in prophylaxis against tuberculosis (TB), a debilitating and deadly infectious disease that infects approximately one-third of the world population. MPLA-modified liposomes loaded with a fusion protein www.sciencedirect.com
Nanoparticles as synthetic vaccines Smith, Morton and Ulery 221
comprised of multiple TB epitopes induced strong T cellmediated immune responses in mice [30]. This is a particularly exciting result since previous TB vaccines have facilitated strong B cell-mediated immune responses which have been found minimally efficacious at preventing subsequent TB infections. A next generation liposome technology that has been utilized for vaccination is the virosome. Virosomes are liposomes that display viral proteins (e.g. influenza-derived hemagglutinin and neuraminidase) on their surface which allow for them to actively fuse with target cells [31]. These complex drug delivery systems possess the versatility of liposomes with highly specific targeting capabilities. Virosomes have been utilized clinically for a variety of prophylactic applications including as tetanus and hepatitis B vaccines [31]. Recently, it has been discovered that virosomal vaccines can be designed to preferentially activate cytotoxic T cells to improve vaccine-based protection against influenza-infected host cells [32].
Virus-like particles Similar to virosomes are a class of biomolecular nanoparticles termed virus-like-particles (VLPs). The inherent difference between the two is that virosomes possess a small concentration of certain viral proteins presented on the surface of liposomes whereas VLPs resemble the traditional structure of viruses (i.e. complete envelope or capsid) except they do not possess any viral genetic material making them non-infectious and incapable of replicating. VLPs can be formed from either one or a combination of viral proteins which can be rapidly engineered to counter pathogenic outbreaks [33,34]. VLPs work well as synthetic vaccines because they possess an external viral shell which the body immediately recognizes as foreign. Quick and effective processing of VLPs yields a rapid and durable host immune response [35]. The first VLP-based human vaccine conveys protection against hepatitis B and has been widely administered since its release in 1986 [36]. More recently VLPs have been utilized as delivery systems for multiple Human Papillomavirus vaccines (HPV) including those marketed as Gardasil and Cervarix. Recent research has shown that VLP-based HPV vaccines induce a highly cross-protective antibody response that conveys protection against a vast majority of HPV serotypes making VLPs robustly effective [37]. Cytotoxic T cells play a key role in clearing infected cells and controlling pathogen load during chronic infection. By targeting this T cell response, VLPs can be very effectively utilized in vaccines.
Micelles While liposomes and VLPs have hydrophilic cores, another group of biomolecular nanoparticles termed micelles possess a hydrophobic core. Micelles form when amphiphilic molecules are solubilized and self-assemble www.sciencedirect.com
by shielding their hydrophobic moieties in their core and displaying the hydrophilic moieties on their surface [38]. Micelles are commonly utilized to deliver poorly water soluble drugs encapsulated in their cores or entrapped amphiphilic molecules [39]. Micelles have been utilized as vaccine delivery systems via two common methods. First, protein vaccines can be easily tethered covalently to the hydrophilic micelle corona. This method has been utilized to attach HIV vaccines to adjuvant-loaded polymer-based micelles yielding strong antigen presenting cell activation in vitro [40]. A similar strategy has been employed for model peptide antigen attached to adjuvant-loaded polymerlipid micelles which were shown to preferentially bind albumin and traffic to the lymph node where the greatly enhanced T cell priming and functioned as an anti-cancer vaccine [41]. Alternatively, peptide amphiphiles can be synthesized by covalently attaching hydrophilic peptide vaccines to hydrophobic moieties (i.e. lipids or aliphatic hydrocarbons) which will self-assemble into micelles in water. By utilizing the vaccine as part of the nanoparticle, peptide amphiphile micelles can achieve antigenic payloads of up to 90% by weight [42]. Peptide amphiphile micelles have been found to induce strong anti-tumor cytotoxic T cell responses and antibody responses against Streptococcus pyogenes in mice without the need for adjuvant supplementation [43,44]. Micelle modulatory provides a unique platform to enhance their capacity as synthetics vaccines. By choosing unique hydrophobic core chemistries, polymer-based micelles can be used to trigger endosome disruption which allows for vaccines to be delivered to the cytosol of cells which yielded antigenic cross-presentation and strong cytotoxic T cell responses in vivo [45]. Also, different amphiphiles with the same or similar hydrophobic moieties are able to self-assemble into heterogeneous micelles. This effect has been leveraged to create peptide amphiphile micelles capable of delivering multiple antigens to the same cell as demonstrated for vaccines against the Herpes simplex virus [46]. Immunostimulating complexes
Similar to micelles, immunostimulating complexes (ISCOMs) are self-assembled structures comprised of biomolecular components (i.e. cholesterols, phospholipids, and Quil A saponins), but self-assembly of the components facilitates the formation of cage-like structures that can be used to entrap adjuvants instead of the hydrophobic core found in micelles. The immunostimulatory function of ISCOMs is due to the fact that many of the heterogeneous mixtures possess Quil A saponins which are known potent adjuvants. While ISCOMs have been studied for nearly 30 years and have been shown quite potent as synthetic vaccine [47], they can induce substantial, undesirable injection-site reactions [48] Current Opinion in Biotechnology 2015, 34:217–224
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which have relegated their development almost exclusively to animal vaccines [49].
4.
A new iteration of ISCOMs termed ISCOMATRIX is similar to the classical ISCOM except the Quil A saponins are first purified to yield a defined group of saponins that do not induce significant inflammation nor toxic side effects [50]. Recent research has shown that ISCOMATRIX can induce both antibody-mediated and cytotoxicmediated immune responses [51] as well as induce HIVspecific immune responses in mice and rabbits [52].
5.
Barhate G, Gautam M, Gairola S, Jadhav S, Pokharkar V: Enhanced mucosal immune responses against tetanus toxoid using novel delivery system comprised of chitosanfunctionalized gold nanoparticles and botanical adjuvant: characterization, immunogenicity, and stability assessment. J Pharm Sci 2014, 11:3448-3456. This in vitro study highlights the combination of two traditionally used biomaterials — chitosan and gold, which when formed into a composite nanparticle greatly enhanced the immune response. 6.
Gregory AE, Judy BM, Qazi O, Blumentritt CA, Brown KA, Shaw AM, Torres AG, Titball RW: A gold nanoparticle-linked glycoconjugate vaccine against Burkholderia mallei. Nanomedicine 2015, 11:447-456.
7.
Guo HC, Feng XM, Sun SQ, Wei YQ, Sun DH, Liu XT, Liu ZX, Luo JX, Yin H: Immunization of mice by hollow mesoporous silica nanoparticles as carriers of porcine circovirus type 2 ORF2 protein. Virol J 2012, 9:108.
8.
Wibowo N, Chuan YP, Seth A, Cordoba Y, Lua LH, Middelberg AP: Co-administration of non-carrier nanoparticles boosts antigen immune response without requiring protein conjugation. Vaccine 2014, 32:3664-3669.
9.
Temchura VV, Kozlova D, Sokolova V, Uberla K, Epple M: Targeting and activation of antigen-specific B-cells by calcium phosphate nanoparticles loaded with protein antigen. Biomaterials 2014, 35:6098-6105.
Conclusions and outlook Over the past decade, significant advancements in synthetic organic chemistry, device fabrication, and molecular-based targeting have let to the generation of a wide range of NPs that can be utilized for a variety of biomedical applications. No field is this more apparent than in vaccinology where inorganic, polymeric, and biomolecular NPs have all been shown to improve weakly immunogenic antigens like peptides and proteins. NP-based synthetic vaccines for prophylaxis against pathogenic infections are currently being motivated into the clinic. While impressive, new applications of NPs as therapeutic and tolerogenic vaccines in the treatment of cancer and autoimmune diseases, respectively, provide even greater possibility for this synthetic vaccine platform. While some generalized fears regarding the implications and toxicity of NPs still remain in the general population, research described in this review supplemented by an archive of studies demonstrating NP biocompatibility and utility in biomedical applications is changing the public perception for the better which will allow for wide adoption of NP-based medical technologies.
Acknowledgements LM thanks the Discovery Fellows Program at the University of Missouri for their support. BDU gratefully acknowledges support from start-up funds from the University of Missouri.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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