Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. DOI 10.1007/s40011-012-0073-6

REVIEW

Nanotechnology in Vaccine Development Amulya K. Panda

Received: 4 August 2011 / Accepted: 4 August 2012 Ó The National Academy of Sciences, India 2012

Abstract Immunization using vaccine has been very successful in combating infectious diseases for more than 200 years now. With understanding of immunology and advancements in molecular biology, it has been possible to develop newer and more efficacious vaccines. However, the new generation vaccines lack the adjuvant and immunostimulatory properties of live/attenuated vaccines. In such scenario, nanotechnology based formulations offer numerous advantages for vaccine developments. Nanocarriers such as liposomes, polymeric particles, virosomes, lipid nanoparticles etc. help in improving the immunogenicity of new generation vaccines. These nanocarrier systems protect the vaccines from degradation, improves its stability, provide adjuvant effect and help in targeting of the antigen to the antigen presenting cells (APCs). It is possible to elicit innate, humoral, cellular or mucosal immune response from antigen formulated with nanocarrier systems. These nanoparticle based systems also offer the advantages of administration in various possible routes. Among the nanocarrier systems, polymer particles have been most widely explored for vaccine developments. Notable among them are particles made from biodegradable polymers of synthetic and natural origin. These polymeric nanoparticles offer numerous advantages for vaccine developments. Formulation of single dose vaccine, elicitation of T cell response, modulation of immune response and mucosal immunity are a few applications of nanotechnology based vaccine formulation. Activation of dendritic cells, vaccination against allergy, tumor immunotherapy and use of polymer particles as an artificial A. K. Panda (&) Staff Scientist VII, Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India e-mail: [email protected]

APCs open new possibilities in controlling infection, cancer and other complex diseases. Keywords Nanoparticles
Adjuvant
Vaccine
Immune response
T cell vaccine
Dendritic cell based vaccine
Immunotherapy

Introduction Vaccine is one of the most important creation of modern medicine providing protection to many infectious diseases [1, 2]. Smallpox has been eradicated [3], polio is on the verge of elimination and many other infections diseases are under control due to use of effective vaccines [4]. Most of the successful vaccines have been developed using empirical observations and work on generation of neutralizing antibody [5]. The duration of protection conferred by vaccines depends mostly on B cell memory responses, however the persistence of antibody titer is often crucial for combating infections. Purified antigens and subunit vaccine have several limitations. The major drawbacks being low level protection requiring repeated immunization, failure to induce cellular response and its inability to work against different serotypes [6, 7]. Vaccine consists of an antigenic unit, adjuvant and a delivery system, thus it is necessary to explore system or technology which could provide adjuvant and delivery system to the new generation vaccines [8]. As the use of vaccine enters the fourth century, it faces numerous challenges to offer protection to many complex infectious diseases [9, 10]. The problems associated with conventional vaccines have led to exploration of new delivery systems and use of adjuvant to tackle problems of new generation vaccines [11, 12]. Among the delivery systems developed, polymer particles have attracted more

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

attention as they offer multiple advantages in eliciting protective immune response to a particular pathogen [13– 15]. Particle based vaccine delivery system offers many solutions to the existing problems of modern vaccines [16, 17]. These delivery systems are typically synthetic carrier systems where the antigen/vaccine of interest is entrapped and delivered to the right antigen presenting cells (APCs) using a normal route of immunization. These particles apart from protecting the antigenic component of the vaccine provide adjuvant effect and offer controlled delivery of antigen [11, 18, 19]. The adjuvant effect of the particulate system is mostly due to preferred uptake by APCs, continuous supply of the antigen from biodegradable particle and activation of the innate immune systems. This can be achieved either by making particles from material which mimic pathogen associated molecular pattern or co-entrapping molecules which activate either innate or adaptive immune system. Polymer particle based vaccine delivery system offers possibilities of delivering the antigen into different compartment of the APC there by providing the possibilities of electing either MHC I or MHC II type immune response [20, 21]. Delivery of antigen using polymer particle based formulation thus offers many opportunities in fine tuning of the immune responses to a particular pathogen or candidate vaccine antigen. Polymer particle based delivery system has been mostly explored for developing single dose vaccine for many diseases [12, 22–24]. However, with the development of nanotechnology, the scope and application on vaccine developments has widened considerably. Nanotechnology as defined by Bawa [25] is defined as the ‘‘design, characterization, production, and application of structure, devices, and systems by controlled manipulation of size and shape at nanometer scale that produce structures, device and systems with at least one novel/superior characteristics and property’’ [26]. Delivery of drugs, vaccines and therapeutics is one of the major applications of nanotechnology where systems like nanoparticles, nanoemulsion, liposomes, polymer micelles, dendrimers etc. provide higher therapeutic indices [26–28]. These nanocarrier systems have the capacity to enter cells and deliver required amount of antigen inside the desired APCs [29]. Further, adjuvant activity can be built in these systems for improved immune responses. This can be achieved either by co-entrapping immunomodulator along with the antigen or surface engineering the particles to activate both innate and adaptive immune system. Nanotechnology based particulate system entrapping candidate vaccine along with adjuvant/immunomodulator, dendritic cell activator thus provides tremendous opportunity for vaccine development. Particularly for the treatment of intracellular infection where both T cell response and B cell mediated antibody responses are required for protective immunity, immunizations with

123

polymeric nanosystem provide a viable solution. Apart from this, nanoparticle based delivery systems can be used for genetic immunization as well as for co-delivery of antigen with immunomodulator and toll like receptors (TLRs). With the success of vaccination for many infectious diseases, currently vaccine strategies are being proposed for allergy, cancer, inflammatory disease and contraception. In the present scenario, it will be very useful to use nanotechnology based vaccine delivery system. Amongst polymer particle based nano delivery systems, formulation based on poly glycolide-co-lactide (PLGA) and poly lactide (PLA) polymers have attracted considerable attraction [14]. It is because these polymers are in human use for a long period of time and are biodegradable. PLGA/ PLA nanoparticles systems are stable in comparison to others, offer higher load of antigen and can be surface modified to reach particular target. Nanoparticle based delivery system using PLA/PLGA polymer thus provide a technology platform for vaccine development [30]. Nanoparticle based vaccine delivery systems and their applications are the focus of this chapter. Formulation of nanocarrier delivery system and their evaluation for eliciting different types of immune response has been discussed. Innovative uses of nanoparticle based delivery system to activate innate, humoral, cellular and mucosal immune response have been described in details. The objective is to highlight the recent developments of particle based delivery system for improved immunogenicity of candidate vaccine antigens. Most of the examples are based on research reports using nanoparticle based formulations using PLGA/ PLA polymer.

Nanotechnology Based Antigen Delivery Systems Classical live attenuated or inactivated pathogen based vaccines are of particulate nature. Even the most successful vaccines like tetanus and diphtheria are made particulate by using alum as an adjuvant. Particulate delivery systems offer many advantages as they are approximately of same size with many pathogens that the immune system is equipped to attack. With the development of drug delivery systems in submicron and micron level for various biomedical applications such systems were explored to improve the immunogenicity of many newer candidate vaccines [26–29]. Since then, many particulate delivery system has been developed based on lipid emulsion, biodegradable polymer, virus like particles (VLP), virosomes and immunostimulatory complex [28–31]. These particulate formulations, apart from delivering the antigen to the target site act as an adjuvant or help in stimulating the immune system. Extensive research has been carried out in recent days on nanoparticle based vaccine delivery system

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

as they provide numerous opportunities for vaccine development. Most notable advantages of nanoparticle based vaccine delivery system have been its ability to deliver the antigen inside the cell leading to T cell response. This is most important, as there is no successful vaccine for intracellular infections where both humoral and cellular response is needed for complete protection. It is thus essential to understand how these nanoparticle systems are designed to deliver the antigen to APCs for improved presentation and processing. Different nanocarrier systems have interesting characteristics which make them appropriate adjuvant for development of new generation vaccines. These nanocarrier systems effectively increase the antigen concentrations in the APCs, mostly in a controlled manner over a long period of time and provide immunostimulatory activity. Various materials have been used to produce nanocarrier systems [28, 32, 33]. These include lipids, polymers, protein, carbohydrates, metals and non-infectious live viruses and bacteria. It is essential that the material used in nanocarrier systems should be non-reactive, biocompatible and biodegradable. Apart from this, the nanocarrier system must have good capacity to carry optimal antigen load to the desired location. It should be stable and have the potential for delivery via various administration routes. This is particularly important as mode of antigen delivery influences the immune response. Based on these above parameters, various nanocarrier systems have been developed for vaccine delivery. As many successful vaccines are composed of live attenuated or dead pathogens, their dummy counterparts have also been used for vaccine delivery systems. These include viral vector based vaccine, bacterial ghost and VLP. Various nanocarrier systems used for vaccine development are described below in brief [26–29]. Viral-Vector Based Antigen Delivery System Viral vectors consist of both replicating and a non-replicating viruses that deliver the vaccine antigen to target cells to induce immune response [34]. Most of the time vaccine antigen is the genetic material from the pathogen against which immunity is desired. Since the immune system has evolved to respond to viruses, viral vector based vaccine using a virus provide the most ideal way to deliver vaccine antigen [28]. Many viruses like vaccinia virus, adenoviruses, alpha viruses have been successfully used to develop delivery system for vaccines [35]. Among all the viral vector based vaccines, adenoviruses have been most extensively used as delivery platform for many vaccines. Safety and efficacy of adenovirus based vaccine delivery have been shown in humans using intranasal and epicutaneous route [36]. Advantages of virally-vectored vaccines include their ease of production and ability to produce both

humoral and cellular response. These viral vectors can be used for nasal, epicutaneous delivery or mucosal immunization and has been reported to improve the immunogenicity of DNA vaccines. Immune response generated by a virally vectored vaccine is further increased by the use of prime boost approach where the primary immunization is carried out by DNA followed by boosting with protein antigen [37]. Such prime boost immunization modality has resulted in elicitation of strong T cell response in many cases [37–39]. Generation of antibody response along with strong T cell response make this prime boost approach most suitable for development of vaccine against intracellular infection. VLPs, Virosomes and Bacterial Ghosts VLPs are non-infective viruses consisting of self-assembled viral envelope proteins without the genetic material [40]. They mimic certain virus properties like size and conformation which help them to elicit strong immune response. Virosomes are liposomes like structure using viral coat protein where envelope of one virus is used as a platform [28]. To this viral coat, components of the virus or another virus or pathogen are attached or entrapped [28, 41]. Both VLPs and virosomes have morphology and cellpenetrating ability similar to an infective viral particle to trigger the immune system. Both the types of particles have been shown to elicit both cellular and humoral immunity [42, 43]. VLPs are generally produced in vitro by transfecting a cell line with plasmid encoding only the viral structural protein followed by entrapment of the candidate vaccine antigen. Recombinant Hepatitis B vaccine and the human papiloma virus vaccine are the two successful examples of VLP based vaccines [44]. VLPs are easy to make and can be produced either in bacteria, plant, insect, animal and yeast cells, however the insect cell system has been most widely used to produce VLPs [45]. VLPs provide improved immune response in comparison to that achieved from vector based as well as virosome based immunization. Because of these reasons, VLP and virosome based vaccine delivery system are currently under evaluation for the development of influenza vaccine [46, 47]. Another system which is being used to delivery antigen to cell is the bacterial ghost system where the nonliving bacterial cell without the genetic component is used for immunization carrying the protein antigen [48]. Like VLPs, bacterial ghosts are made up of nonliving envelope of the cells having native antigenic structure including its bio-adhesive properties. Escherichia coli, Salmonella typhimurium, BCG, V. cholerae and many other gram negative bacteria have been used as bacterial ghost for gene delivery [49]. Bacterial cell walls promote T cell activation, induces systemic, cellular and mucosal immunity to

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

target antigen and thus provide a model platform for vaccine delivery. Immunostimulating Complexes (ISCOMs) The ISCOMs are novel vaccine delivery vehicles with potent adjuvant activity [50]. ISCOMs are lipid particles comprising of cholesterol, phospholipids and protein antigens formulated by incorporating saponin Quil A from the soapbark tree Quillaja saponaria [51]. These are cage like structures having size around 40–50 nm. Antigens are trapped within ISCOMs through apolar interaction [52]. ISCOMs are described as a novel structures for presentation of membrane proteins from a viruses to APCs to elicit immune response [53]. These particles due to their lipophilic nature are preferentially taken up by APCs such as DCs, monocytes and macrophages. Immune responses observed by ISCOMs are mostly due to antigen presentation by both MHC class I and class II pathways resulting in elicitation of both cellular and humoral immune response [54, 55]. Immunomodulatory capability of the saponin helps in further augmentation of immune response using ISCOMs. Smaller size of ISCOMs and its negative surface ensures colloidal nature of the adjuvant and thus make them very stable preparation for vaccine delivery purpose. ISCOMATRIX is another delivery system without antigen [55]. Antigen is added to the iscomatrix during formulation for improved immunogenicity. When administered in mice, ISCOMs elicit strong mucosal, systemic as well as CTL response [52]. Even though ISCOMs have been used for improved immunogenicity of different antigen, toxicity of saponin is one of the major concerns for human application. Currently ISCOMs and ISCOMATRIX based delivery systems are only registered for veterinary applications. Liposomes Liposomes are spherical vesicular systems composed of a phospholipids bilayer shell with an aqueous core extensively used for drug delivery [56]. Phospholipids found in mammalians cells are mostly used for the preparation of liposomes as they are non-toxic. Liposomes can be fabricated as unillamelar vesicles as small as 20 nm or as multilamellar vesicles as large as 50 l. Liposomal delivery of amphotericin B and doxorubicin are the most important successful nanodelivery systems [27]. In liposomes, the antigen can either be in the core of the liposome, buried within the lipid bilayer or can be adsorbed on the liposomal surface [57, 58]. Both antigen and adjuvant can also be co-delivered using liposomes [33, 59]. Encapsulations of the antigens in the core of the liposome protect the antigen from degradation and thus help in improving its

123

immunogenicity. Antigen adsorbed on liposomal surface can be directly used for antigen presentations to APCs. Immunostimulatory properties of liposomes are mostly due to their ability to release antigen over a prolonged period of time with preferential internalization by APCs. Liposomes have been used to improve both humoral and cellular immune response [60]. The versatility of liposomes lies in the fact that these can easily be surface modified to do diverse functions such as extended circulation and intracellular delivery. Based on these properties, liposomes have been mainly classified as conventional liposome, pH sensitive liposome, cationic liposome, immunoliposome and long circulating liposomes [59]. These liposomes have different types of antigen release profile which may be suitable for a particular type of vaccine depending upon the required immune response. Immunoliposomes and long circulating immunoliposome promote targeted delivery of antigen to APCs and improve immune response. Another interesting application of liposomes has been intracellular delivery of peptides particularly using cell penetrating peptide for vaccine delivery [59, 61]. Cell penetrating peptide based liposome have the capability to present the antigen both to MHC I and MHC II pathways and thus provide a powerful tool for vaccine delivery. Liposome incorporated/attached with dendritic cell activating substances provides opportunity for induction of immune response through dendritic cell activation [62]. Surface modified liposome with chitosan derivatives have been successfully used to deliver DNA vaccine for elicitation of both humoral and cellular response [63]. Muramyl dipeptide (MDP) co-entrapped with hepatitis B surface antigen in liposome formulation have been reported to improve the immunogenicity of the entrapped antigen [64]. With many success stories particularly for delivery of drugs and anticancer agents, liposome delivery of antigen for vaccine development hold great promise for the future [60]. As the liposome have the capabilities to entrap both antigen and immunomodulatory agent, they have high potential to contribute towards the development of effective vaccine formulations. Polymeric Nanoparticles Polymeric particles are well established system for the delivery of vaccines [17, 65]. These are essentially solid particles made of polymers either derived from natural source or from synthetic origin entrapping/encapsulating the candidate antigen. These particles offer advantages in terms of size, load and release profile of antigen and thus are preferred over other carrier systems. The most important role of polymeric nanoparticles in terms of vaccine

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

development are, modulation of immune response, enhanced uptake of antigen, targeting to particular APCs depending on size and surface chemistry and more importantly protecting the antigen inside the polymer matrix for a longer period of time in vivo. Because of these advantages, polymeric nanoparticle based formulations have been extensively explored for vaccine development. A variety of polymers have been used for nanoparticle formulations for drug/vaccine delivery. However, the most commonly studied polymers are poly D L lactide-co-glycolide (PLGA) and polylactide (PLA) [13, 14, 66]. These biodegradable, biocompatible polymers have been approved by FDA for use in humans and have been extensively used for development of single dose vaccine delivery system [17, 24, 67]. In polymeric particle formulations, antigen are either entrapped or adsorbed to the surface of the polymer particles. These can act as a depot from which antigen is released in a controlled manner. Most of these polymers induce inflammation thus provide adjuvant effect. Apart from this, polymeric nanoparticles have the capability to elicit innate immune response. Multiple antigen delivery in the same particle and codelivery of antigen and adjuvant can also be achieved with polymeric nanocarrier systems. Chitosan has been widely used for delivery of antigens [68, 69]. Because of its bio-adhesiveness, it has been tried mostly for mucosal delivery of antigens [70]. Different sized particles with varying load of antigen could be formulated for the delivery of antigen to achieve desired immune response. Highly cationic charges of chitosan make it particularly suitable for genetic immunization as formulation of particle with plasmid DNA is easily achieved through electrostatic attraction. Varieties of nanocarrier systems can be formulated using chitosan based formulation for immunization [33]. These include chitosan coated PLGA particles, electrostatic chitosan DNA complexes, nanoparticles made from chitosan and its derivatives and finally chitosan coated nanoemulsion. These coatings are particularly suitable for mucosal delivery of vaccine due to adhesive properties of the chitosan [70]. Nanoparticles made from amphiphillic poly amino acid derivates have also been reported for delivery of antigens [71]. Polyglutamic acid based nanoparticles have been extensively used for delivery of antigens [72–74]. Using polyglutamic acid based nanoparticle systems; both humoral and cellular response has been observed. Polyglutamic acid based nanoparticles have been used as delivery system for AIDS vaccine as well as a tumor vaccine [72, 73]. Polyethylenimine amine (PEI) based nanoparticle system have been successfully used for delivery of nucleic acid [75, 76]. Efficient transfection ability of such polymeric nanosystem provided a viable alternative for genetic immunization to combat many intracellular infections.

Polymeric Micelles Polymeric micelles are spherical colloidal particles formed by non-covalent binding of amphiphillic polymers [27, 77]. For the formation of micelles, amphiphillic molecules must have both hydrophobic and hydrophilic segments which form particles with a hydrophobic interior and a hydrophilic exterior. In the core of the micelles water-insoluble drugs are solubilized whereas water soluble molecules are adsorbed at the surface. Molecule or drugs with intermediate polarity are distributed along with the surfactant molecules in between surface and inner core. As the size of polymeric micelles is less than 50 nm in diameter, these are mostly suitable for intravenous applications. Polymeric micelles are mostly stable and have long circulation time in blood. This favours the use of polymeric micelles for controlled release of drugs as well as for in vivo imaging by entrapping contrast agent within the hydrophobic core or by linking covalently to the surface of micelles. Polymeric micelles are mostly used for delivery of insoluble drugs but have been recently explored for vaccine delivery purpose. Polymeric micelles using combination of polylactic acid and polyethylene glycol has been reported for the delivery of hepatitis B vaccine [78]. Micelles formed using tri-block polymer induced better humoral antibody response. Polymer micelles have also been used for maturation of dendritic cells [79]. In near future such nanocarrier systems using micelles from tri-block polymer will be exploited for vaccine delivery. Dendrimers Dendrimers are a class of polymeric macromolecules characterized by branched three dimensional structures around the central core through repeated branching [80]. These are formed via divergent or convergent synthesis by a series of controlled polymerization reactions [81]. This results in the formation of highly branched spherical macromolecules analogous to a globular protein. Dendrimers are generally generated from monomers where the number of units is more than 2–3 polymerizing from a central core [81]. Dendrimers resemble like spheres where they entrap molecules in the cavities of the branched structure and are usually 10–100 nm in diameter with multiple functional groups on their surface. Drugs are loaded either by complexation or formation of chemical bond at terminal branch points. The most noted examples of dendrimers is polyamidoamine (PAMAM) which is synthesized by the repetitive addition of branching units to ammonia or ethylene diamine. Because of its size and polyvalent nature, a dendrimer has the capacity to activate multiple receptors simultaneously which results in new or improved biological actions [82]. A novel mannose based

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

antigen delivery system using PAMAM based dendrimers have been reported recently [83]. It was observed that mannosylated dendrimers based ovalbumin was a potent immune inducer and helped in inducing potent CD4/CD8 T cell and antibody response. It is expected that with several advantages and excellent adjuvant property, mannosylated dendrimers will be a potential vaccine carrier. Tetragallyo lysine based dendrimers (TGDK) have been reported to deliver antigen to M cells [84]. Oral administration of antigen conjugated to TGDK elicited significantly higher IgA response. Apart from delivering antigen, dendrimers based nanocarrier is an excellent vehicle for transfection studies [85]. With the scope of modifying the branched chain in dendrimers, it is expected that they will be more useful for developing targeted vaccine delivery system. Solid Nanoparticles (SNPs) are Vaccine Carrier SNPs are made of biodegradable materials, such as proteins, fats, lipids, polystyrene and other polymers and are the most commonly used nanocarrier systems [86]. These particles may be solid lipid nanoparticles (SLNs), liposphers and particles made from polystyrene and other materials. Ranging in size from 10 to 1,000 nm, SNPs can be used simultaneously for imaging and drug delivery. A major advantage of these particles is that they release the entrapped drug in a controlled manner. Rapamune is the first nanoparticle-mediated medicine used as an immunosuppressant to prevent organ transplant rejection approved by US FDA in 2000. Albumin nanoparticle entrapping paclitaxel is the most successful nanoformulations. SLNs because of their physical stability and controlled release characteristics is an excellent drug delivery platform [87]. SLNs because of their versatility and stability can be administered by most of the routes generally used for drug delivery [88]. Topical application using SLN is already in the market for therapeutic and cosmetic application [89]. SLNs have been used for vaccine delivery system as they release the antigen in controlled manner and provide adjuvant effect [21, 86]. Lipid particles have been reported to trigger internalization of BSA by the APCs [90]. Solid lipid nanoparticles have been explored for the delivery of Hepatitis B vaccine [91]. Mucosal immunization of HBsAg loaded SLNs has been reported to elicit higher IgA response.

recombinant protein based antigens and subunit vaccines lack all the required components of live or attenuated vaccines. In such situation, adjuvants and delivery system complement to elicit effective immune response from the candidate vaccines [8]. Polymer particles made from natural and synthetic polymer release the antigen in controlled manner and provide additional adjuvant effect. Polymer such as chitosan, albumin, polyesters, polyanhydride and more recently polyamino acid based polymers have been extensively used for vaccine delivery purpose. Biodegradable polymer particle based vaccine delivery systems have the capabilities to mimic multi-dose vaccination schedule from a single immunization dose. Thus for many infectious diseases where neutralizing antibody titers provide protective immunity, polymer particles serve as an ideal delivery platform [14, 22]. Polymers such as polylactide (PLA) and polylactide-co-glycolide (PLGA) have been widely explored for development of single dose vaccine formulation [13, 16, 65]. Apart from safety (biocompatible, biodegradable and Food and Drug Administration approved polymers for human use) and stability of the entrapped antigens, vaccine delivery using PLA/PLGA polymeric particulate systems offers possibility of use as single dose applications for generation of immune response. These polymers are not toxic and metabolized into lactic acid and glycolic acid which are degraded by normal route. The degradation product is safe and has no toxicity issue for human uses. There are numerous ways by which polymer particles of different sizes can be formulated using normal pharmaceutical formulation technology [92]. In fact, half a dozen drug delivery formulations using PLGA/PLA based polymer are already in the market [93]. Major advantages of PLA/PLGA polymer based particulate vaccine delivery system include: (1) (2) (3)

(4)

(5) Immunogenicity of Polymer Particle Based Vaccine Formulation

(6) (7)

The ultimate objective of vaccination is to offer long term protection by eliciting effective immune response. This is achieved by inducing specific T cell and memory B cells to produce circulating antibodies. Purified antigens,

123

(8)

Generation of long lasting immune response from single dose. Protection of the entrapped protein antigens in vivo. Controlled or modified rate of release profile of entrapped antigen during formulation by altering various formulation parameters. Entrapment of multiple antigens in same particle or/ and co-entrapment of antigen, adjuvant and or immunomodulator. Possibility of administering vaccine through oral or nasal mucosa. Targeted delivery of entrapped antigen with surface modification or on the basis of size. Modulation of immune response—humoral or cellmediated immune response based on size of the particles. Enhancement of immunological memory using polymer particle entrapped antigens.

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

Because of the above advantages PLGA/PLA polymer particle based vaccine delivery systems have been extensively explored in multiple ways to improve the immunogenicity of candidate vaccines. Single Dose Vaccine using Polymeric Particles Poly lactide-co-glycolide (PLGA) and polylactide (PLA) polymer particles entrapping vaccine/antigens as single dose application provide an alternative to the multi-dose vaccine injection schedule for immunization [22]. These polymeric formulations release the antigen from the particle either continuously or in pulsatile manner to mimic the multi-dose immunization schedule generally used for many vaccines to achieve protective immunity. Polymer particles, particularly made from PLGA/PLA, not only work as a delivery system but also provide adjuvant activity [13, 17]. These polymeric particles based delivery system have the capacity to activate both humoral and/or cellular response [94, 95]. Efficient targeting of antigens to APCs using polymer particles has been considered to be the main factor for improving the immunogenicity of the entrapped antigens. To elicit desired antibody response from polymer particles, it is essential that these polymer particles protect the immunoreactive antigens during formulation and releases the entrapped antigens mimicking the conventional vaccination schedule [96]. A further enhancement in immune response using polymer particle based system can be achieved by using additional adjuvants like alum or immunomodulator along with the polymer particles. This strategy has been adopted for a number of candidate antigens such as Diphtheria toxoid (DT) and Hepatitis B surface antigen (HBsAg) [13, 97, 98]. Enhanced antibody response has been reported while immunizing with admixture of alum and biodegradable nanoparticles containing tetanus toxoid (TT) [99]. Immunization using admixture of polymer particle entrapped antigen and alum resulted in improved antibody titer from single point immunization [100, 101]. Immunogenicity of candidate peptide vaccine entrapped in PLGA polymer particle has also been reported to be enhanced considerably in comparison to the soluble antigens [102]. Particularly for diseases where multiple injections are required such as vaccination against rabies and hepatitis B infection, polymer particle entrapping antigen will be an ideal system to achieve immunity from single point immunizations.

antibody formation is mostly through class II pathways. For presentation of antigen through class I pathway, it is essential that the antigen be present inside the cytosol. Since most of the current vaccines fail to elicit potent CTL response, they are ineffective against intracellular infection. Moreover, in some situation both T cell and B cell responses are needed in controlled way to combat infection. In such a situation, polymeric particle based delivery system is very effective. It has been observed that polymeric nanoparticle because of their potential to enter APCs has the capacity to deliver the antigen inside the cells. This activates class I presentation pathways leading to T cell activation. Numerous proteins and peptides have been delivered intracellularly and CTL responses have been generated using PLGA polymer based nanoparticle system [103–105]. Four types of vaccine formulations have the potential to elicit CD8/CTlL response. These are live attenuated vaccine, live vector vaccine, DNA vaccine and heterologous prime boost vaccine [106]. In all the cases, vaccine antigen is delivered intracellularly and continuously present for a longer period of time. It is expected that polymeric nanoparticles will mimic the above characteristics of live attenuated vaccines and will elicit T cell response. Two mechanisms have been proposed to explain the ability of nanoparticle system to produce T cell response. The first is the escape of the nanoparticle to the cytoplasm by disrupting the phagosomes where the released antigen is presented as endogenous protein. The load of the protein and the release characteristics decide the strength of T cell response. The other mechanism is the delivery of particles to phagolysosome compartments that contain MHC I receptor that are being recycled from plasma membrane. Proteins are degraded by phagolysosome enzymes into antigenic peptides that complex MHC I receptor and are then trafficked to the cell surface for presentation. Thus nanoparticle system has the ability to process the exogenous antigen in class I presentation pathways leading to T cell responses. Size, surface properties of the nanoparticles, their charge and chemical composition are important for elicitation of antigen specific cytotoxic T cell response [72, 105]. Activation of T cell response using polymeric particle is the most exciting application of nanotechnology based vaccine formulation. Modulation of Immune Response using Polymeric Nanoparticles

T Cell Response using Polymeric Nanoparticles Activation of cytotoxic T lymphocytes (CTLs) is essential for the development of immunity against viruses, intracellular infections and tumors. APCs activate CTLs through class I antigen presentation pathways whereas

It has been reported by many researchers that particle size influences the quality of immune response [107–109]. Particles in submicron and micron ranges elicit both type 1 and type 2 immune response [94]. It was observed that HBsAg entrapped microparticles elicited higher antibody

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

titer than nanoparticles and admixture of particles and alum improved the antibody titers considerably [110]. Immunization with microparticles and alum (MP ? alum) elicited antibody titers better than from alum adsorbed HBsAg. Similar variations in antibody titer were also observed for TT, thus indicating that nanoparticles elicit lower antibody response where as microparticles generate higher antibody response [110]. To know how different sized polymeric delivery systems decide the quality of immune response, antibody isotypes have been analyzed [110]. It was also observed that, immunization with microparticles resulted in higher IgG1/IgG2a ratio than nanoparticles based immunization. Similar sized microparticles which give rise to higher antibody response promote IL-4 secretion; where as nanoparticles favor IFN-c secretion [110]. This indicated that antigens are processed and presented differently from nanoparticles in comparison to microparticles. Thus using different sized particles, antigens can be delivered through different routes into the APCs and will generate different types of immune response. This was in concurrence with the concept that microparticles promote humoral response where as nanoparticles promote cellular response. These results indicated that apart from controlled release applications, these polymeric particulate formulations have wide variety of applications in vaccine design. Nanoparticles alone or mixture of nanoparticles and microparticles can be used to elicit predominantly cellular or a mixed cellular and humoral response using the same antigen. This may be very useful for the treatment of intracellular infection where both cytotoxic T cell activation and antibody production provide protective immunity particularly to treat intracellular infections [111]. Use of polymer particles has also been reported for modulation of innate and adaptive immune response [112]. Particle size, particularly at nanoscale range has been reported to enhance interferon secretion where as microparticles induced tumor necrosis factor-a in human cells [113]. Admixture of nanoparticle and micro-particle have shown to elicit sustained antibody titers from single dose intramuscular immunization of TT particles [100]. These results highlight the importance of particle size on modulation of immune response and provide an opportunity to use nanoparticles to elicit desired immune response. Cross Presentation of Exogenous Antigen using Polymeric Nanoparticle In general, intracellular antigens are presented through major histocompatibility complex (MHC) class I molecules where as external antigens are processed through phagocytic compartment using MHC class II molecules. However in some cases external antigens can be presented on

123

MHC class I molecule to stimulate CTL (cytotoxic T lymphocyte) response. This process is called cross presentation and is important for generation of CTL response [114, 115]. This pathway helps in acquisition of antigen from infected, or component of dead cells by APCs and facilitate the generation of MHC class I restricted immune response. The most important requirement for cross presentation is the escape of antigen from phagosome to cytosol; however phagosome alone is also competent for cross presentation of exogenous antigen [116]. Generation of CTL response through cross presentation would be an ideal strategy for vaccine formulation for many intracellular infections where T cell response is required for protection. However the efficiency of cross presentation of exogenous antigen by APCs is very low. This limit’s the exploitation of cross presentation pathways for elicitation of CTL response for protein antigens. In such situation, protein antigens entrapped in biodegradable polymeric nanoparticle provide a real advancement for cross presentation of exogenous antigen to elicit CTL response. These polymeric nanoparticles have the capacity to deliver the antigen in cytosol as they escape the endosome. PLGA nanoparticles have been shown to improve the efficiency of cross presentation in DCs, B cells and macrophages [117, 118]. Entrapment of antigen in PLGA particles not only delivers the antigen in the cytosol but also activates the MHC class I presentation more than 100-fold in comparison to the soluble antigen. Entrapped antigen in PLGA particle also elicit higher amount of IL-2 both in DCs and B cells. It was also observed that the MHC presentation is TAP depended and these particles serve as intracellular reservoir for sustained delivery and presentation of antigen through MHC class I pathways. Polymer particle size also influences the degree of cross presentation of exogenous antigens [119]. This suggests that nanoparticle based vaccine delivery system has capabilities to elicit CTL response particularly by presenting the antigen through cross presentation pathways. Mucosal Immune Response using Polymeric Nanoparticle The specialized immune system of the mucosal surface is due to the mucosa associated lymphoid tissue (MALT) which promotes both innate and adaptive immune response [120]. The MALT involves aero-digestive, the uro-genital tracts, eye conjunctiva, ducts of all exocrine gland and the internal ear. MALT has functionally distinct B cells, T cells and accessory cell population to elicit immune response. As the mucosal surface represents a major site of entry for many pathogens, mucosal vaccine delivery provides a realistic approach for achieving protective immunity. Around half-a dozen of vaccines are immunized through

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

mucosal route to provide protection and the notable among them being cholera vaccine, polio vaccines, typhoid vaccine and rotavirus vaccine [32]. Although IgA is the predominant humoral defense mechanism at mucosal surfaces, locally produced IgM and IgG also contributes towards the development of protective immunity. Generation of cytotoxic T lymphocyte response has been reported after mucosal immunization using various routes. The success story of Flu mist (intranasal vaccine by Medimmune Vaccines Inc. USA, www.mediumune.com) highlights the importance of mucosal immunization for combating diseases. One of the major problems of mucosal vaccinations is the relatively poor immune response achieved after immunization [121]. This is mostly due to compartmentalization of the MALT, lower uptake of antigen due to mucosal barrier and degradation of protein antigen by the protease of the mucosal surface. Because of this compartmentalization, only oral and nasal delivery systems have been explored for mucosal vaccination [33]. Oral and nasal vaccinations are easy as they avoid use of needles and have better compliance. However, poor immunogenic response is the major problem of mucosal vaccine formulations. In such situation nanotechnology based vaccine formulations provide a viable delivery platform for mucosal immunization [32]. Entrapment of candidate vaccine in polymeric nanoparticle has been shown to offer many advantages for mucosal vaccination [122]. It protects the antigen from protease attack, help in penetration of mucosal membrane and thus presents the antigen to APCs [123]. Nanoparticle based mucosal vaccine formulation interacts with APCs to promote better presentation of the antigen to generate both cellular and humoral response [124]. PLGA nanoparticles with surface coating of lectins given orally elicited both mucosal and systemic immune response [125, 126]. To protect the antigen from acidic microenvironment of stomach, nanoparticle using tri-block polymers have been used to orally deliver hepatitis B vaccine [127]. Nanoparticles using tri-block polymer not only protects the antigen from degradation but also results in generation of improved immune response from single point immunization. Mucosal vaccine using prime boost approach and nanogel based mucosal delivery system are the most exciting nanoparticle based adjuvant free mucosal vaccine delivery systems [14, 128]. Apart from delivering the antigen through mucosal route, use of additional adjuvant provide further improvement in eliciting mucosal based immune response using nanocarrier system. Thus by careful consideration of carrier system, co-entrapment of mucosa based adjuvant along with antigen, choice of target tissue and mode of delivery, it will be possible to design an efficient mucosal vaccine delivery system.

Novel Applications of Antigen Loaded Nanoparticle Systems Initially developed as controlled release vaccine delivery system, antigen loaded polymeric nanoparticles have shown promises in other related areas. The capacity to present the immunogenic antigen/protein to different cellular system offers varieties of possibilities for treatment of diseases. Activation of lymphatic system [129], delivery of drugs and candidate vaccine using aerodynamic polymer particles [29] and more importantly use of fluorescence’s labeled polymer nanoparticle as diagnostic tools [130] are a few of the other applications of nanotechnology based antigen formulations. More than activating immune response to elicit desired immunity, nanoparticles have been found to be very useful in many unconventional situations. The capacity to deliver and release the antigen in controlled manner has many more applications in modern medicine. Polymeric particle can be loaded with multiple drugs, combination of drugs and antigens or for coentrapment of different vaccine representing various serotypes of pathogen provide an exciting opportunity. In the following section some novel applications related to nanotechnology based immune intervention has been described. It is expected that in near future more emphasis and probably more clinical out come will appear from this non-classical use of nanoparticle based vaccine delivery system. Antigen Delivery Through Dendritic Cells (DCs) DCs are important APCs that connect innate and adaptive immune response [131]. DCs are distributed throughout the body for the detection of foreign pathogen which they ingest and deliver it at the lymph node for T cell activation [131]. They have the capability to activate naive T cells. This is mostly due to their ability to recognize foreign pathogen and danger signal, expression of MHC class I and class II and other co-stimulatory molecules that are required for efficient presentation of antigens for T cell activation [132]. DCs function in three sequential steps. Firstly it captures antigen and helps in its migration to lymphoid organs; then it promotes maturation of DCs for antigen processing and finally helps in secretion of costimulatory molecules and cytokines for presentation of antigen either through MHC class I or MHC class II pathways. Because of these multifarious capabilities, DCs are considered to the most efficient APCs. Activation of DCs using nanoparticle based antigen delivery system thus provides tremendous potential for developing improved vaccine formulation [133]. PLGA and PLA based polymeric particle entrapping candidate antigens have been extensively explored for

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

delivery of antigens to DCs [132]. Nanoparticles are passively taken up by DCs and deliver the antigen in non specific ways. Using 100–200 nm particles, DCs in lymph nodes have been targeted and enhanced immune response has been observed [109]. PLGA/PLA based polymer particles have excellent ability to carry antigen load and release them in controlled manner but has the disadvantages that they do not recognize DCs or activate DCs [134]. However, maturation of DCs has been reported by surface coating/co-entrapment of TLR ligands on polymer particles [135]. Most recently it has been reported that size of the particle influences innate immune response through danger signals [113]. With surface modification and co-entrapment of ligands, the capability of PLGA particle to activate DCs has been enhanced significantly. Conjugation of polymeric particle using DCs specific antibody, peptide or lectin has been successfully used to target the nanoparticle to DCs [136, 137]. Dendritic cell maturation and selective presentations of antigen through MHC class I/II pathways has also been reported using polymer particle co-entrapping TLR ligands [138, 139]. Lipopolysaccharide incorporating nanoparticles have been reported to activate inflammasome [140]. Nanoparticle based vaccine delivery system has the capability to use different pathways to activate innate and adaptive immune system. Targeting nanoparticle containing antigen load to DCs thus provide a promising strategy for inducing protective immune response through T cell activation.

However the most important achievement using polymeric particles for allergen delivery will be elicitation of T cell response. It has been reported in many cases that PLGA/PLA based nanoparticles elicit preferentially Th1 response. This will be most beneficial for allergen specific immunotherapy where entrapping the antigen in polymer particles will preferentially induces Th1 type immune response [146]. This shift the allergen induced immune response from pathological Th2 response to therapeutic Th1 response [144, 146]. Shifting of typical Th2 response induced by birch pollen allergen to Th1 response has been observed using PLGA particle based immunizations [147, 148]. It was also observed that entrapment of birch allergen in PLGA particle induced IgG2a but not IgG1 type antibody indicating the suitability of polymer particle based oral immunization for allergen therapy. Recently protective allergy vaccine while co-entrapping CpG motifs along with allergen has been reported [149]. Entrapment of venom phospholipase along with CpG in PLGA particle has been shown to induce protective IgG2a response and lower IgG response. Delivery for allergen using polymeric particles thus provide compliance for vaccination, reduce the duration of treatment and more importantly have the potentials to modulate the allergic immune response. Nanoparticle based vaccine delivery system for allergen specific immunotherapy will be very useful in near future.

Use of Polymeric Particles for Development of Vaccine Against Allergy

Nanoparticle based vaccine delivery systems have the capacity to deliver the antigen intracellularly thus they can be used for eliciting immune response against cancer through generation of cell mediated immune response. This involves antigen specific lymphocyte priming in vivo or stimulation and expansion of antigen specific T cells ex vivo and its subsequent retransfer into patients. The former process is called active immunotherapy where as the later is know as adoptive immune therapy. Success of these cancer therapies requires expansion of antigen specific T cells [150, 151]. In such scenario, polymer particle improves the performances related to both active and adoptive immunotherapy. Entrapment of the cancer antigen in polymer particles not only improve its immunogenicity but also activate T cell response to the cancer antigens [152]. Cancer antigen entrapped in nanoparticles helps in introducing the antigen in an immunogenic form, either to break tolerance or to activate the T cell repertoire [153]. This helps in generation of CD8 T cell response against tumor and helps in clearing them. Numerous cancer antigens have been entrapped in polymer particles and have shown improved performances for cancer treatment. These nanoparticles deliver the antigens to APCs with high efficiency; induce effectors T cell response for tumor

About one-fourth of the population in developed countries have allergy which is mostly due to Ig E mediated type 1 hypersensitivity [141]. The clinical manifestation of such allergy are hay fever, asthma, eczema or even anaphylactic reaction. Subcutaneous allergen specific immunotherapy (SIT) is the only therapy use to desensitize the patients [142]. During SIT, gradual increasing doses of allergen is injected which shift the immune response against the allergen from Th2 type response to Th1 type immune response [143, 144]. SIT has two major side effects (i) allergic side effect and (ii) it needs 30–80 injections over the years with increasing doses to achieve effective desensitization. In such situation biodegradable polymeric particle are most suitable vaccine delivery systems for generation of allergen specific immune response. These particles have the capacity to release the antigen over a period of time and thus reduce the burden of multiple injections to achieve complete sensitization. More importantly, as the allergen is entrapped in side the polymer particle it is not exposed to either mast cells or basophil cells to induce undesirable side effect [145].

123

Nanoparticle as a Tool for Immunotherapy of Cancer

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

clearance [153]. Even cancer antigens along with TLR ligands co-entrapped in same polymer particles have been reported to elicit potent CD8 T cell mediated anti- tumor immunity [152]. Polyethylenimine (PEI) based nanoparticles have been successfully used for tumor targeting and tumor gene therapy [154, 155]. Chondroitin and hyaluronic acid based composite PEI particles have been used to deliver DNA to solid tumors thus opening an exciting possibility of tumor targeting using nanoparticle based system. Polymeric Particle as Artificial APC Polymeric nanoparticles have been recently exploited as a vehicle for adoptive immunotherapy where lymphocytes are expanded in vivo and then infused back to the body for treatment. The most important requirement for T cell activation is the requirement of APCs. Expenses and time involved in isolation and culturing of quality APCs for T cell activation is very high. Because of these reasons, many investigators have started using artificial antigen presenting cells (aAPCs). aAPCs can be cell based or polymer particle based [156, 157]. Recently polymer particle have attracted more attention for the development of aAPCs. This is because all necessary signals required for T cell activation such as reorganization signal, co-stimulatory signal and more importantly controlled release of cytokine for T cell activation can be provided from polymer particle mimicking an APCs. Liposome formulation, magnetic bead and PLGA based particles have been successfully used as artificial APCs. Among these, PLGA polymer based particles provide the best T cell stimulation because they provide all the necessary signals for T cell activation. Expansion of functional T cell using artificial APC made from polymer particle have been reported [158, 159]. More than 40-fold enhancement of T cell expansion was achieved using PLGA particle based aAPCs [159]. Polymeric particle based formulations thus help in improving the ex vivo T cell expansion. These results thus provide a very novel use of polymeric particles for improved immunotherapy particularly for treatment of cancer and intracellular infection where activated T cells are required to achieve desired immunity.

Future Prospects Vaccines provide the most wonderful strategy to activate immune response to combat infection. Most successful vaccines work by generation of neutralizing antibody through adaptive immune response. However many intracellular pathogen and cancer treatment require activation of cytotoxic T cell response to combat infection. Normal vaccine which consists of live/attenuated or subunits of

antigens fails to elicit T cell response. Apart from this, many times it is necessary to activate the innate immune response along with adoptive response to protect infection. In these scenarios, nanoparticle based vaccine delivery system provides a viable solution and offer tremendous opportunity to fine tune immune response. Many recombinant protein candidate vaccines are poorly immunogenic and have stability problems. This is taken care by nanoparticle based delivery system. Apart from this, nanoparticle based delivery system has the ability to deliver antigen at particular site to promote antigen presentation and processing according to the need. Modulation of immune response and generation of cytotoxic T cell response is the most attractive benefits of nanoparticle based vaccine delivery system. It is expected that novel candidate vaccine particularly for malaria and tuberculosis with the aid of nanotechnology based delivery system will be able to elicit both humoral and cellular response which are necessary to provide immunity for combating intracellular infections. Nanotechnology based formulations are being investigated as vaccine carriers, adjuvant, and drug delivery system to target infection. Liposomes, polymeric particle based system has shown novel applications in terms of development of allergy vaccine, mucosal vaccine and most importantly for adoptive immunotherapy. The use of nanoparticle system as aAPCs provided an exciting area of research particularly for cancer immunotherapy. Some polymeric nanoparticle based formulations are in clinical developments for infectious diseases. Although understanding of nanoparticle and its use on activating immune system have been explored fairly, molecular interaction of these particles with cell [160] toxicity, safety [161] and more importantly regulatory issue need detail exploration. Pharmacokinetics and anatomical distribution of nanoparticle based vaccine delivery system and its interaction with all types of APC need careful analysis [162–164]. Finally, there is need to establish production of such nanoparticle based antigen delivery systems using good manufacturing practices (GMP). Very little is reported on clinical grade manufacturing of nanoparticle based vaccine delivery systems. This needs careful monitoring to produce reproducible batches of nanoparticle based stable delivery systems. Once these issues are addressed, the full potential of this technology can be realized and be put to use for human welfare. Pathogens continuously develop a number of strategies to evade the immune system to cause infection. In such scenario delivering vaccine with nanotechnology offer counter attack strategy to combat infection [29]. It is expected that with the advancement of nanotechnology, new generation of vaccine formulation will be developed for controlling infection, cancer, metabolic and complex diseases.

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. Acknowledgments Thanks to director National Institute of Immunology, New Delhi for supporting the research activities on vaccine delivery using polymeric particles. AKP is partly supported by TATA Innovation Fellowship of the Dept. of Biotechnology, Govt. of India.

References 1. Hilleman MR (2000) Vaccines in historic evolution and perspective: a narrative of vaccine discoveries. J Hum Virol 3:63–76 2. Ada G (2001) Vaccines and vaccination. N Engl J Med 345:1042–1053 3. Andre FE (2003) Vaccinology: past achievements, present roadblocks and future promises. Vaccine 21:593–595 4. Plotkin SA (2005) Vaccines: past, present and future. Nat Med 11:S5–S11 5. Lambert PH, Liu M, Siegrist CA (2005) Can successful vaccines teach us how to induce efficient protective immune responses? Nat Med 11:S54–S62 6. Hilleman MR (2002) Overview of the needs and realities for developing new and improved vaccines in the 21st century. Intervirology 45:199–211 7. Rappuoli R (2007) Bridging the knowledge gaps in vaccine design. Nat Biotechnol 25:1361–1366 8. O’Hagan DT, Valiante NM (2003) Recent advances in the discovery and delivery of vaccine adjuvants. Nat Rev Drug Discov 2:727–735 9. Clemens J, Jodar L (2005) Introducing new vaccines into developing countries: obstacles, opportunities and complexities. Nat Med 11:S12–S15 10. Plotkin SA (2009) Vaccines: the fourth century. Clin Vaccine Immunol 16:1709–1719 11. Guy B (2007) The perfect mix: recent progress in adjuvant research. Nat Rev Microbiol 5:505–517 12. Harandi AM, Medaglini D, Shattock RJ (2010) Vaccine adjuvants: a priority for vaccine research. Vaccine 28:2363–2366 13. Gupta RK, Chang AC, Siber GR (1998) Biodegradable polymer microspheres as vaccine adjuvants and delivery systems. Dev Biol Stand 92:63–78 14. Jiang W, Gupta RK, Deshpande MC, Schwendeman SP (2005) Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens. Adv Drug Deliv Rev 57:391–410 15. Rice-Ficht AC, Arenas-Gamboa AM, Kahl-McDonagh MM, Ficht TA (2010) Polymeric particles in vaccine delivery. Curr Opin Microbiol 13:106–112 16. Storni T, Kundig TM, Senti G, Johansen P (2005) Immunity in response to particulate antigen-delivery systems. Adv Drug Deliv Rev 57:333–355 17. O’Hagan DT, Singh M, Ulmer JB (2006) Microparticle-based technologies for vaccines. Methods 40:10–19 18. Aguilar JC, Rodriguez EG (2007) Vaccine adjuvants revisited. Vaccine 25:3752–3762 19. Jones KS (2008) Biomaterials as vaccine adjuvants. Biotechnol Prog 24:807–814 20. Men Y, Audran R, Thomasin C, Eberl G, Demotz S, Merkle HP, Gander B, Corradin G (1999) MHC class I- and class IIrestricted processing and presentation of microencapsulated antigens. Vaccine 17:1047–1056 21. Eyles JE, Carpenter ZC, Alpar HO, Williamson ED (2003) Immunological aspects of polymer microsphere vaccine delivery systems. J Drug Target 11:509–514 22. Cleland JL (1999) Single-administration vaccines: controlledrelease technology to mimic repeated immunizations. Trends Biotechnol 17:25–29

123

23. Singh M, Srivastava I (2003) Advances in vaccine adjuvants for infectious diseases. Curr HIV Res 1:309–320 24. Singh M, Chakrapani A, O’Hagan D (2007) Nanoparticles and microparticles as vaccine-delivery systems. Expert Rev Vaccines 6:797–808 25. Bawa R (2007) Patents and nanomedicine. Nanomedicine 2:351–374 26. Bawarski WE, Chidlowsky E, Bharali DJ, Mousa SA (2008) Emerging nanopharmaceuticals. Nanomedicine 4:273–282 27. Xiang SD, Scholzen A, Minigo G, David C, Apostolopoulos V, Mottram PL, Plebanski M (2006) Pathogen recognition and development of particulate vaccines: does size matter. Methods 40:1–9 28. Peek LJ, Middaugh CR, Berkland C (2008) Nanotechnology in vaccine delivery. Adv Drug Deliv Rev 60:915–928 29. Look M, Bandyopadhyay A, Blum JS, Fahmy TM (2010) Application of nanotechnologies for improved immune response against infectious diseases in the developing world. Adv Drug Deliv Rev 62:378–393 30. Lu JM, Wang X, Marin-Muller C, Wang H, Lin PH, Yao Q, Chen C (2009) Current advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev Mol Diagn 9:325–341 31. Couvreur P, Vauthier C (2006) Nanotechnology: intelligent design to treat complex disease. Pharm Res 23:1417–1450 32. Chadwick S, Kriegel C, Amiji M (2010) Nanotechnology solutions for mucosal immunization. Adv Drug Deliv Rev 62:394–407 33. Csaba N, Garcia-Fuentes M, Alonso MJ (2009) Nanoparticles for nasal vaccination. Adv Drug Deliv Rev 61:140–157 34. Liniger M, Zuniga A, Naim HY (2007) Use of viral vectors for the development of vaccines. Expert Rev Vaccines 6:255–266 35. Robert-Guroff M (2007) Replicating and non-replicating viral vectors for vaccine development. Curr Opin Biotechnol 18:546–556 36. Barouch DH, Nabel GJ (2005) Adenovirus vector-based vaccines for human immunodeficiency virus type 1. Hum Gene Ther 16:149–156 37. Draper SJ, Heeney JL (2010) Viruses as vaccine vectors for infectious diseases and cancer. Nat Rev Microbiol 8:62–73 38. Moore AC, Hill AV (2004) Progress in DNA-based heterologous prime-boost immunization strategies for malaria. Immunol Rev 199:126–143 39. Liu J, O’Brien KL, Lynch DM, Simmons NL, La Porte A, Riggs AM, Abbink P, Coffey RT, Grandpre LE, Seaman MS, Landucci G, Forthal DN, Montefiori DC, Carville A, Mansfield KG, Havenga MJ, Pau MG, Goudsmit J, Barouch DH (2009) Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 457:87–91 40. Roy P, Noad R (2008) Virus-like particles as a vaccine delivery system: myths and facts. Hum Vaccine 4:5–12 41. Gluck R, Burri KG, Metcalfe I (2005) Adjuvant and antigen delivery properties of virosomes. Curr Drug Deliv 2:395–400 42. Huckriede A, Bungener L, Stegmann T, Daemen T, Medema J, Palache AM, Wilschut J (2005) The virosome concept for influenza vaccines. Vaccine 23(Suppl 1):S26–S38 43. Grgacic EV, Anderson DA (2006) Virus-like particles: passport to immune recognition. Methods 40:60–65 44. Buckland BC (2005) The process development challenge for a new vaccine. Nat Med 11:S16–S19 45. Krammer F, Grabherr R (2010) Alternative influenza vaccines made by insect cells. Trends Mol Med 16:313–320 46. Wilschut J (2009) Influenza vaccines: the virosome concept. Immunol Lett 122:118–121 47. Kang SM, Song JM, Quan FS, Compans RW (2009) Influenza vaccines based on virus-like particles. Virus Res 143:140–146

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 48. Mayr UB, Walcher P, Azimpour C, Riedmann E, Haller C, Lubitz W (2005) Bacterial ghosts as antigen delivery vehicles. Adv Drug Deliv Rev 57:1381–1391 49. Seow Y, Wood MJ (2009) Biological gene delivery vehicles: beyond viral vectors. Mol Ther 17:767–777 50. Cox JC, Sjolander A, Barr IG (1998) ISCOMs and other saponin based adjuvants. Adv Drug Deliv Rev 32:247–271 51. Skene CD, Sutton P (2006) Saponin-adjuvanted particulate vaccines for clinical use. Methods 40:53–59 52. Sun HX, Xie Y, Ye YP (2009) ISCOMs and ISCOMATRIX. Vaccine 27:4388–4401 53. Morein B, Sundquist B, Hoglund S, Dalsgaard K, Osterhaus A (1984) ISCOM, a novel structure for antigenic presentation of membrane proteins from enveloped viruses. Nature 308:457–460 54. Hu KF, Lovgren-Bengtsson K, Morein B (2001) Immunostimulating complexes (ISCOMs) for nasal vaccination. Adv Drug Deliv Rev 51:149–159 55. Pearse MJ, Drane D (2005) ISCOMATRIX adjuvant for antigen delivery. Adv Drug Deliv Rev 57:465–474 56. Torchilin VP (2005) Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 4:145–160 57. Alving CR (1991) Liposomes as carriers of antigens and adjuvants. J Immunol Methods 140:1–13 58. Alving CR (1995) Liposomal vaccines: clinical status and immunological presentation for humoral and cellular immunity. Ann N Y Acad Sci 754:143–152 59. Torchilin V (2009) Multifunctional and stimuli-sensitive pharmaceutical nanocarriers. Eur J Pharm Biopharm 71:431–444 60. Altin JG, Parish CR (2006) Liposomal vaccines-targeting the delivery of antigen. Methods 40:39–52 61. Brooks NA, Pouniotis DS, Tang CK, Apostolopoulos V, Pietersz GA (2010) Cell-penetrating peptides: application in vaccine delivery. Biochim Biophys Acta 1805:25–34 62. Foged C, Arigita C, Sundblad A, Jiskoot W, Storm G, Frokjaer S (2004) Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition. Vaccine 22:1903–1913 63. Khatri K, Goyal AK, Gupta PN, Mishra N, Mehta A, Vyas SP (2008) Surface modified liposomes for nasal delivery of DNA vaccine. Vaccine 26:2225–2233 64. Jain V, Vyas SP, Kohli DV (2009) Well-defined and potent liposomal hepatitis B vaccines adjuvanted with lipophilic MDP derivatives. Nanomedicine 5:334–344 65. Langer R, Cleland JL, Hanes J (1997) New advances in microsphere-based single-dose vaccines. Adv Drug Deliv Rev 28:97–119 66. Johansen P, Men Y, Merkle HP, Gander B (2000) Revisiting PLA/PLGA microspheres: an analysis of their potential in parenteral vaccination. Eur J Pharm Biopharm 50:129–146 67. Waeckerle-Men Y, Groettrup M (2005) PLGA microspheres for improved antigen delivery to dendritic cells as cellular vaccines. Adv Drug Deliv Rev 57:475–482 68. Illum L, Jabbal-Gill I, Hinchcliffe M, Fisher AN, Davis SS (2001) Chitosan as a novel nasal delivery system for vaccines. Adv Drug Deliv Rev 51:81–96 69. Kang ML, Cho CS, Yoo HS (2009) Application of chitosan microspheres for nasal delivery of vaccines. Biotechnol Adv 27:857–865 70. Alpar HO, Somavarapu S, Atuah KN, Bramwell VW (2005) Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery. Adv Drug Deliv Rev 57:411–430 71. Akagi T, Wang X, Uto T, Baba M, Akashi M (2007) Protein direct delivery to dendritic cells using nanoparticles based on amphiphilic poly(amino acid) derivatives. Biomaterials 28:3427–3436 72. Yoshikawa T, Okada N, Oda A, Matsuo K, Matsuo K, Kayamuro H, Ishii Y, Yoshinaga T, Akagi T, Akashi M, Nakagawa

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85. 86.

87. 88.

89. 90.

S (2008) Nanoparticles built by self-assembly of amphiphilic gamma-PGA can deliver antigens to antigen-presenting cells with high efficiency: a new tumor-vaccine carrier for eliciting effector T cells. Vaccine 26:1303–1313 Wang X, Uto T, Akagi T, Akashi M, Baba M (2008) Poly(gamma-glutamic acid) nanoparticles as an efficient antigen delivery and adjuvant system: potential for an AIDS vaccine. J Med Virol 80:11–19 Uto T, Wang X, Sato K, Haraguchi M, Akagi T, Akashi M, Baba M (2007) Targeting of antigen to dendritic cells with poly(gamma-glutamic acid) nanoparticles induces antigen-specific humoral and cellular immunity. J Immunol 178:2979–2986 Swami A, Goyal R, Tripathi SK, Singh N, Katiyar N, Mishra AK, Gupta KC (2009) Effect of homo bifunctional crosslinkers on nucleic acids delivery ability of PEI nanoparticles. Int J Pharm 374:125–138 Patnaik S, Arif M, Pathak A, Kurupati R, Singh Y, Gupta KC (2010) Cross-linked polyethylenimine-hexametaphosphate nanoparticles to deliver nucleic acids therapeutics. Nanomedicine 6:344–354 Kwon GS (2003) Polymeric micelles for delivery of poorly water-soluble compounds. Crit Rev Ther Drug Carrier Syst 20:357–403 Jain AK, Goyal AK, Gupta PN, Khatri K, Mishra N, Mehta A, Mangal S, Vyas SP (2009) Synthesis, characterization and evaluation of novel triblock copolymer based nanoparticles for vaccine delivery against hepatitis B. J Control Release 136:161–169 Boudier A, Aubert-Pouessel A, Louis-Plence P, Gerardin C, Jorgensen C, Devoisselle JM, Begu S (2009) The control of dendritic cell maturation by pH-sensitive polyion complex micelles. Biomaterials 30:233–241 Patri AK, Majoros IJ, Baker JR (2002) Dendritic polymer macromolecular carriers for drug delivery. Curr Opin Chem Biol 6:466–471 Zeng F, Zimmerman SC (1997) Dendrimers in supramolecular chemistry: from molecular recognition to self-assembly. Chem Rev 97:1681–1712 Samad A, Alam MI, Saxena K (2009) Dendrimers: a class of polymers in the nanotechnology for the delivery of active pharmaceuticals. Curr Pharm Des 15:2958–2969 Sheng KC, Kalkanidis M, Pouniotis DS, Esparon S, Tang CK, Apostolopoulos V, Pietersz GA (2008) Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo. Eur J Immunol 38:424–436 Misumi S, Masuyama M, Takamune N, Nakayama D, Mitsumata R, Matsumoto H, Urata N, Takahashi Y, Muneoka A, Sukamoto T, Fukuzaki K, Shoji S (2009) Targeted delivery of immunogen to primate m cells with tetragalloyl lysine dendrimer. J Immunol 182:6061–6070 Shcharbin DG, Klajnert B, Bryszewska M (2009) Dendrimers in gene transfection. Biochemistry (Mosc) 74:1070–1079 Almeida AJ, Souto E (2007) Solid lipid nanoparticles as a drug delivery system for peptides and proteins. Adv Drug Deliv Rev 59:478–490 Joshi MD, Muller RH (2009) Lipid nanoparticles for parenteral delivery of actives. Eur J Pharm Biopharm 71:161–172 Muller RH, Mader K, Gohla S (2000) Solid lipid nanoparticles (SLN) for controlled drug delivery: a review of the state of the art. Eur J Pharm Biopharm 50:161–177 Souto EB, Muller RH (2005) SLN and NLC for topical delivery of ketoconazole. J Microencapsul 22:501–510 Erni C, Suard C, Freitas S, Dreher D, Merkle HP, Walter E (2002) Evaluation of cationic solid lipid microparticles as synthetic carriers for the targeted delivery of macromolecules to phagocytic antigen-presenting cells. Biomaterials 23:4667–4676

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 91. Saraf S, Mishra D, Asthana A, Jain R, Singh S, Jain NK (2006) Lipid microparticles for mucosal immunization against hepatitis B. Vaccine 24:45–56 92. Jain RA (2000) The manufacturing techniques of various drug loaded biodegradable poly (lactide-co-glycolide) (PLGA) devices. Biomaterials 21:2475–2490 93. Chaubal M (2002) Polylactide/Glycolides: excipients for injectable drug delivery & beyond. Drug Deliv Technol 2: 34–36 94. Mottram PL, Leong D, Crimeen-Irwin B, Gloster S, Xiang SD, Meanger J, Ghildyal R, Vardaxis N, Plebanski M (2007) Type 1 and 2 immunity following vaccination is influenced by nanoparticle size: formulation of a model vaccine for respiratory syncytial virus. Mol Pharm 4:73–84 95. Ataman-Onal Y, Munier S, Ganee A, Terrat C, Durand PY, Battail N, Martinon F, Le Grand R, Charles MH, Delair T, Verrier B (2006) Surfactant-free anionic PLA nanoparticles coated with HIV-1 p24 protein induced enhanced cellular and humoral immune responses in various animal models. J Control Release 112:175–185 96. Raghuvanshi RS, Singh Om, Panda AK (2001) Formulation and characterization of immunoreactive tetanus toxoid biodegradable polymer particles. Drug Deliv 9:839–843 97. Singh M, Li XM, Wang H, McGee JP, Zamb T, Koff W, Wang CY, O’Hagan DT (1998) Controlled release microparticles as a single dose diphtheria toxoid vaccine: immunogenicity in small animal models. Vaccine 16:346–352 98. Shi L, Caulfield MJ, Chern RT, Wilson RA, Sanyal G, Volkin DB (2002) Pharmaceutical and immunological evaluation of a single-shot hepatitis B vaccine formulated with PLGA microspheres. J Pharm Sci 91:1019–1035 99. Raghuvanshi RS, Katare YK, Lalwani K, Ali MM, Singh O, Panda AK (2002) Improved immune response from biodegradable polymer particles entrapping tetanus toxoid by use of different immunization protocol and adjuvants. Int J Pharm 245:109–121 100. Katare YK, Panda AK, Lalwani K, Haque IU, Ali MM (2003) Potentiation of immune response from polymer-entrapped antigen: toward development of single dose tetanus toxoid vaccine. Drug Deliv 10:231–238 101. Katare YK, Muthukumaran T, Panda AK (2005) Influence of particle size, antigen load, dose and additional adjuvant on the immune response from antigen loaded PLA microparticles. Int J Pharm 301:149–160 102. Kirby DJ, Rosenkrands I, Agger EM, Andersen P, Coombes AG, Perrie Y (2008) PLGA microspheres for the delivery of a novel subunit TB vaccine. J Drug Target 16:282–293 103. Murthy N, Xu M, Schuck S, Kunisawa J, Shastri N, Frechet JM (2003) A macromolecular delivery vehicle for protein-based vaccines: acid-degradable protein-loaded microgels. Proc Natl Acad Sci USA 100:4995–5000 104. Lu D, Garcia-Contreras L, Xu D, Kurtz SL, Liu J, Braunstein M, McMurray DN, Hickey AJ (2007) Poly (lactide-co-glycolide) microspheres in respirable sizes enhance an in vitro T cell response to recombinant Mycobacterium tuberculosis antigen 85B. Pharm Res 24:1834–1843 105. Shi S, Hickey AJ (2010) PLGA microparticles in respirable sizes enhance an in vitro T cell response to recombinant Mycobacterium tuberculosis antigen TB10.4-Ag85B. Pharm Res 27:350–360 106. Robinson HL, Amara RR (2005) T cell vaccines for microbial infections. Nat Med 11:S25–S32 107. Gutierro I, Hernandez RM, Igartua M, Gascon AR, Pedraz JL (2002) Size dependent immune response after subcutaneous, oral and intranasal administration of BSA loaded nanospheres. Vaccine 21:67–77

123

108. Brewer JM, Pollock KG, Tetley L, Russell DG (2004) Vesicle size influences the trafficking, processing, and presentation of antigens in lipid vesicles. J Immunol 173:6143–6150 109. Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF (2008) Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol 38:1404–1413 110. Kanchan V, Panda AK (2007) Interactions of antigen-loaded polylactide particles with macrophages and their correlation with the immune response. Biomaterials 28:5344–5357 111. Zinkernagel RM (2002) On differences between immunity and immunological memory. Curr Opin Immunol 14:523–536 112. Uto T, Akagi T, Hamasaki T, Akashi M, Baba M (2009) Modulation of innate and adaptive immunity by biodegradable nanoparticles. Immunol Lett 125:46–52 113. Rettig L, Haen SP, Bittermann AG, von Boehmer L, Curioni A, Kramer SD, Knuth A, Pascolo S (2010) Particle size and activation threshold: a new dimension of danger signaling. Blood 115:4533–4541 114. Ackerman AL, Cresswell P (2004) Cellular mechanisms governing cross-presentation of exogenous antigens. Nat Immunol 5:678–684 115. Rock KL, Shen L (2005) Cross-presentation: underlying mechanisms and role in immune surveillance. Immunol Rev 207:166–183 116. Houde M, Bertholet S, Gagnon E, Brunet S, Goyette G, Laplante A, Princiotta MF, Thibault P, Sacks D, Desjardins M (2003) Phagosomes are competent organelles for antigen cross-presentation. Nature 425:402–406 117. Shen H, Ackerman AL, Cody V, Giodini A, Hinson ER, Cresswell P, Edelson RL, Saltzman WM, Hanlon DJ (2006) Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles. Immunology 117:78–88 118. Yang YW, Hsu PY (2008) The effect of poly(D, L-lactide-coglycolide) microparticles with polyelectrolyte self-assembled multilayer surfaces on the cross-presentation of exogenous antigens. Biomaterials 29:2516–2526 119. Tran KK, Shen H (2009) The role of phagosomal pH on the sizedependent efficiency of cross-presentation by dendritic cells. Biomaterials 30:1356–1362 120. Holmgren J, Czerkinsky C (2005) Mucosal immunity and vaccines. Nat Med 11:S45–S53 121. Neutra MR, Kozlowski PA (2006) Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 6:148–158 122. Sharma S, Mukkur TK, Benson HA, Chen Y (2009) Pharmaceutical aspects of intranasal delivery of vaccines using particulate systems. J Pharm Sci 98:812–843 123. Florindo HF, Pandit S, Goncalves LM, Videira M, Alpar O, Almeida AJ (2009) Antibody and cytokine-associated immune responses to S. equi antigens entrapped in PLA nanospheres. Biomaterials 30:5161–5169 124. Yang K, Whalen BJ, Tirabassi RS, Selin LK, Levchenko TS, Torchilin VP, Kislauskis EH, Guberski DL (2008) A DNA vaccine prime followed by a liposome-encapsulated protein boost confers enhanced mucosal immune responses and protection. J Immunol 180:6159–6167 125. Gupta PN, Khatri K, Goyal AK, Mishra N, Vyas SP (2007) M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B. J Drug Target 15:701–713 126. Mishra N, Tiwari S, Vaidya B, Agrawal GP, Vyas SP (2011) Lectin anchored PLGA nanoparticles for oral mucosal immunization against hepatitis B. J Drug Target 19:67–78 127. Jain AK, Goyal AK, Mishra N, Vaidya B, Mangal S, Vyas SP (2010) PEG-PLA-PEG block co-polymeric nanoparticles for oral immunization against hepatitis B. Int J Pharm 387:253–262

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 128. Nochi T, Yuki Y, Takahashi H, Sawada S, Mejima M, Kohda T, Harada N, Kong IG, Sato A, Kataoka N, Tokuhara D, Kurokawa S, Takahashi Y, Tsukada H, Kozaki S, Akiyoshi K, Kiyono H (2010) Nanogel antigenic protein-delivery system for adjuvantfree intranasal vaccines. Nat Mater 9:572–578 129. Reddy ST, van der Vlies AJ, Simeoni E, Angeli V, Randolph GJ, O’Neil CP, Lee LK, Swartz MA, Hubbell JA (2007) Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat Biotechnol 25:1159–1164 130. Hauck TS, Giri S, Gao Y, Chan WC (2010) Nanotechnology diagnostics for infectious diseases prevalent in developing countries. Adv Drug Deliv Rev 62:438–448 131. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K (2000) Immunobiology of dendritic cells. Annu Rev Immunol 18:767–811 132. Reddy ST, Swartz MA, Hubbell JA (2006) Targeting dendritic cells with biomaterials: developing the next generation of vaccines. Trends Immunol 27:573–579 133. Babensee JE (2008) Interactions of dendritic cells with biomaterials. Semin Immunol 20:101–108 134. Fischer S, Uetz-von Allmen E, Waeckerle-Men Y, Groettrup M, Merkle HP, Gander B (2007) The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles. Biomaterials 28:994–1004 135. Wischke C, Zimmermann J, Wessinger B, Schendler A, Borchert HH, Peters JH, Nesselhut T, Lorenzen DR (2009) Poly(I:C) coated PLGA microparticles induce dendritic cell maturation. Int J Pharm 365:61–68 136. Kwon YJ, James E, Shastri N, Frechet JM (2005) In vivo targeting of dendritic cells for activation of cellular immunity using vaccine carriers based on pH-responsive microparticles. Proc Natl Acad Sci USA 102:18264–18268 137. Cruz LJ, Tacken PJ, Fokkink R, Joosten B, Stuart MC, Albericio F, Torensma R, Figdor CG (2010) Targeted PLGA nano but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro. J Control Release 144:118–126 138. Elamanchili P, Diwan M, Cao M, Samuel J (2004) Characterization of poly(D, L-lactic-co-glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells. Vaccine 22:2406–2412 139. Heit A, Schmitz F, Haas T, Busch DH, Wagner H (2007) Antigen co-encapsulated with adjuvants efficiently drive protective T cell immunity. Eur J Immunol 37:2063–2074 140. Demento SL, Eisenbarth SC, Foellmer HG, Platt C, Caplan MJ, Mark SW, Mellman I, Ledizet M, Fikrig E, Flavell RA, Fahmy TM (2009) Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy. Vaccine 27:3013–3021 141. Ring J, Eberlein-Koenig B, Behrendt H (2001) Environmental pollution and allergy. Ann Allergy Asthma Immunol 87:2–6 142. Carnes J, Robinson DS (2008) New strategies for allergen immunotherapy. Recent Pat Inflamm Allergy Drug Discov 2:92–101 143. Larche M (2002) Anti-T-cell strategies in the treatment of allergic disease. Allergy 57(Suppl 72):20–23 144. Rolland JM, Gardner LM, O’Hehir RE (2009) Allergen-related approaches to immunotherapy. Pharmacol Ther 121:273–284 145. Jilek S, Walter E, Merkle HP, Corthesy B (2004) Modulation of allergic responses in mice by using biodegradable poly(lactide-coglycolide) microspheres. J Allergy Clin Immunol 114:943–950 146. Johansen P, Martinez Gomez JM, Gander B (2007) Development of synthetic biodegradable microparticulate vaccines: a roller coaster story. Expert Rev Vaccines 6:471–474 147. Roth-Walter F, Scholl I, Untersmayr E, Ellinger A, Boltz-Nitulescu G, Scheiner O, Gabor F, Jensen-Jarolim E (2005) Mucosal targeting of allergen-loaded microspheres by Aleuria aurantia lectin. Vaccine 23:2703–2710

148. Scholl I, Kopp T, Bohle B, Jensen-Jarolim E (2006) Biodegradable PLGA particles for improved systemic and mucosal treatment of Type I allergy. Immunol Allergy Clin N Am 26:349–364 149. Martinez Gomez JM, Fischer S, Csaba N, Kundig TM, Merkle HP, Gander B, Johansen P (2007) A protective allergy vaccine based on CpG- and protamine-containing PLGA microparticles. Pharm Res 24:1927–1935 150. Ho WY, Blattman JN, Dossett ML, Yee C, Greenberg PD (2003) Adoptive immunotherapy: engineering T cell responses as biologic weapons for tumor mass destruction. Cancer Cell 3:431–437 151. Paulos CM, Suhoski MM, Plesa G, Jiang T, Basu S, Golovina TN, Jiang S, Aqui NA, Powell DJ Jr, Levine BL, Carroll RG, Riley JL, June CH (2008) Adoptive immunotherapy: good habits instilled at youth have long-term benefits. Immunol Res 42:182–196 152. Hamdy S, Molavi O, Ma Z, Haddadi A, Alshamsan A, Gobti Z, Elhasi S, Samuel J, Lavasanifar A (2008) Co-delivery of cancerassociated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8 ? T cell-mediated anti-tumor immunity. Vaccine 26:5046–5057 153. Goforth R, Salem AK, Zhu X, Miles S, Zhang XQ, Lee JH, Sandler AD (2009) Immune stimulatory antigen loaded particles combined with depletion of regulatory T-cells induce potent tumor specific immunity in a mouse model of melanoma. Cancer Immunol Immunother 58:517–530 154. Pathak A, Swami A, Patnaik S, Jain S, Chuttani K, Mishra AK, Vyas SP, Kumar P, Gupta KC (2009) Efficient tumor targeting by polysaccharide decked polyethylenimine based nanocomposites. J Biomed Nanotechnol 5:264–277 155. Pathak A, Kumar P, Chuttani K, Jain S, Mishra AK, Vyas SP, Gupta KC (2009) Gene expression, bio-distribution, and pharmacoscintigraphic evaluation of chondroitin sulfate-PEI nanoconstructs mediated tumor gene therapy. ACS Nano 3:1493– 1505 156. Kim JV, Latouche JB, Riviere I, Sadelain M (2004) The ABCs of artificial antigen presentation. Nat Biotechnol 22:403–410 157. Oelke M, Krueger C, Giuntoli RL, Schneck JP (2005) Artificial antigen-presenting cells: artificial solutions for real diseases. Trends Mol Med 11:412–420 158. Zappasodi R, Di Nicola M, Carlo-Stella C, Mortarini R, Molla A, Vegetti C, Albani S, Anichini A, Gianni AM (2008) The effect of artificial antigen-presenting cells with pre-clustered anti-CD28/-CD3/-LFA-1 monoclonal antibodies on the induction of ex vivo expansion of functional human antitumor T cells. Haematologica 93:1523–1534 159. Steenblock ER, Fahmy TM (2008) A comprehensive platform for ex vivo T-cell expansion based on biodegradable polymeric artificial antigen-presenting cells. Mol Ther 16:765–772 160. Nel AE, Madler L, Velegol D, Xia T, Hoek EM, Somasundaran P, Klaessig F, Castranova V, Thompson M (2009) Understanding bio-physicochemical interactions at the nano-bio interface. Nat Mater 8:543–557 161. Igarashi E (2008) Factors affecting toxicity and efficacy of polymeric nanomedicines. Toxicol Appl Pharmacol 229:121– 134 162. Owens DE III, Peppas NA (2006) Opsonization, bio-distribution, and pharmacokinetics of polymeric nanoparticles. Int J Pharm 307:93–102 163. Li SD, Huang L (2008) Pharmacokinetics and bio-distribution of nanoparticles. Mol Pharm 5:496–504 164. Alexis F, Pridgen E, Molnar LK, Farokhzad OC (2008) Factors affecting the clearance and bio-distribution of polymeric nanoparticles. Mol Pharm 5:505–515

123