Takami Akagi

Targeting of Antigen to Dendritic Cells with Poly(γ-Glutamic Acid) Nanoparticles Induces Antigen-Specific Humoral and Cellular Immunity

Nanoparticles are considered to be efficient tools for inducing potent immune responses by an Ag carrier. In this study, we examined the effect of Ag-carrying biodegradable poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) on the induction of immune responses in mice. The NPs were efficiently taken up by dendritic cells (DCs) and subsequently localized in the lysosomal compartments. γ-PGA NPs strongly induced cytokine production, up-regulation of costimulatory molecules, and the enhancement of T cell stimulatory capacity in DCs. These maturational changes of DCs involved the MyD88-mediated NF-κB signaling pathway. In vivo, γ-PGA NPs were preferentially internalized by APCs (DCs and macrophages) and induced the production of IL-12p40 and IL-6. The immunization of mice with OVA-carrying NPs induced Ag-specific CTL activity and Ag-specific production of IFN-γ in splenocytes as well as potent production of Ag-specific IgG1 and IgG2a Abs in serum. Furthermore, immunization with NPs carrying a CD8+ T cell epitope peptide of Listeria monocytogenes significantly protected the infected mice from death. These results suggest that Ag-carrying γ-PGA NPs are capable of inducing strong cellular and humoral immune responses and might be potentially useful as effective vaccine adjuvants for the therapy of infectious diseases.

Biodegradable Nanoparticles as Vaccine Adjuvants and Delivery Systems: Regulation of Immune Responses by Nanoparticle-Based Vaccine

Polymeric nano- and microparticles have recently been shown to possess significant potential as drug delivery systems. In particular, the use of biodegrad- able polymeric nanoparticles with entrapped antigens such as proteins, peptides, or DNA represents an exciting approach for controlling the release of vaccine antigens and optimizing the desired immune response via selective targeting of the antigen to antigen-presenting cells (APCs). The efficient delivery of antigens to APCs, especially in dendritic cells (DCs), and the activation of APCs are some of the most important issues in the development of effective vaccines. Using nanoparticle- based vaccine delivery systems, it is possible to target delivery to DCs, activate these APCs, and control release of the antigen. Nanoparticles prepared from biodegradable and biocompatible polymers such as poly(lactide-co-glycolide) (PLGA), poly(amino acid)s, and polysaccharides have been shown to be effective

Nanoparticles built by self-assembly of amphiphilic γ-PGA can deliver antigens to antigen-presenting cells with high efficiency : A new tumor-vaccine carrier for eliciting effector T cells

Nanotechnology is a fundamental technology for designing and generating innovative carriers for biomacromolecular drugs. Biodegradable poly(gamma-glutamic acid)-based nanoparticles (gamma-PGA NPs) are excellent vaccine carriers capable of delivering antigenic proteins to antigen-presenting cells (APCs) and eliciting potent immune responses based on antigen-specific cytotoxic T lymphocytes. In mice, subcutaneous immunization with gamma-PGA NPs entrapping ovalbumin (OVA) more effectively inhibited the growth of OVA-transfected tumors than immunization with OVA emulsified using Freunds complete adjuvant. In addition, gamma-PGA NPs did not induce histopathologic changes after subcutaneous injection or acute toxicity through intravenous injection. Importantly, gamma-PGA NPs efficiently delivered entrapped antigenic proteins into APCs, and these antigen-capturing APCs migrated to regional lymph nodes. Our results demonstrate that a gamma-PGA NP system for antigen delivery will advance the clinical utility of vaccines against cancer.

The induction of innate and adaptive immunity by biodegradable poly(γ-glutamic acid) nanoparticles via a TLR4 and MyD88 signaling pathway

The induction of adaptive immunity through the activation of innate immunity is indispensable for vaccine development. Although strategies for particulate antigen delivery are widely investigated, their immunological mechanisms are unclear. We describe in this study that biodegradable nanoparticles (NPs) elaborated with poly(γ-glutamic acid) (γ-PGA) are able to induce potent innate and adaptive immune responses through Toll-like receptor 4 (TLR4) and MyD88 signaling pathways. The production of inflammatory cytokines from macrophages and the maturation of dendritic cells were impaired in MyD88-knockout and TLR4-deficient mice compared with their wild-types, when the cells were stimulated with γ-PGA NPs. The immunization of these mice with antigen-carrying γ-PGA NPs also resulted in diminished induction of antigen-specific cellular immune responses. These results suggest that γ-PGA NPs have not only an antigen-carrying capacity but also a potent adjuvant function of eliciting adaptive immune responses to the carrying antigen through recognition of the first-line host-sensor system.

Poly(γ-glutamic acid) nano-particles combined with mucosal influenza virus hemagglutinin vaccine protects against influenza virus infection in mice

Adding poly(gamma-glutamic acid) nano-particles (gamma-PGA-NPs), a safe, natural material, to subcutaneous immunization with influenza virus hemagglutinin (HA) vaccine increases the protective immune responses against influenza virus in mice. Here, we examined whether intranasal administration of the HA vaccine with gamma-PGA-NPs would induce protection from influenza virus challenge in mice. Intranasal immunization with the mixture of gamma-PGA-NPs and HA vaccine from an influenza virus strain A/PR/8/34 (H1N1) or A/New Caledonia/20/99 (H1N1) enhanced protection of mice from A/PR/8/34 infection. Intranasal immunization with A/New Caledonia/20/99 HA vaccine and gamma-PGA-NPs induced cell-mediated immune responses and neutralizing antibody production for both A/New Caledonia/20/99 and A/PR/8/34. These data suggest that gamma-PGA-NPs may have potential for clinical applications as a mucosal adjuvant.

Multifunctional conjugation of proteins on/into bio-nanoparticles prepared by amphiphilic poly(γ-glutamic acid)

The present study focuses on nanoparticles composed of amphiphilic poly(γ-glutamic acid) (γ-PGA) as potential protein carriers. Amphiphilic graft co-polymers composed of γ-PGA as the hydrophilic backbone and L-phenylalanine ethylester (L-PAE) as the hydrophobic segment were synthesized by grafting L-PAE to γ-PGA using water-soluble carbodiimide (WSC). Due to their amphiphilic properties, the γ-PGA-graft-L-PAE co-polymer formed monodispersed nanoparticles in water. The particle size of the nanoparticles composed of γ-PGA-graft-L-PAE (γ-PGA nanoparticles) was about 200 nm and showed a highly negative zeta potential. To evaluate their potential applications as multifunctional protein carrier, we prepared protein-entrapped γ-PGA nanoparticles by encapsulation, covalent immobilization or physical adsorption methods. For this purpose, 11 different proteins with various molecular weights and isoelectric points (pI values) were used as model proteins. The encapsulation of the protein into the nanoparticles was observed for all tested proteins. The amount of protein covalently immobilized or adsorbed onto the nanoparticles showed different tends based on the molecular weight and pI of each protein. Positively charged proteins could be adsorbed onto the negatively charged nanoparticles by electrostatic interaction. Moreover, it was found that enzyme-encapsulated nanoparticles showed higher enzymatic activity than surface-immobilized nanoparticles. These results indicated that the enzymatic activity of the enzyme-entrapped nanoparticles was significantly affected by the conjugation method, and that encapsulation was the optimal method for the conjugation of proteins and nanoparticles. It is expected that the γ-PGA nanoparticle will have great potential as multifunctional carriers in pharmaceutical and biomedical applications, such as drug and vaccine delivery systems.

Intracellular degradation and distribution of protein-encapsulated amphiphilic poly(amino acid) nanoparticles.

Physicochemical properties, such as particle size, shape, molecular weight, surface charge and composition, play a key role in the cellular uptake of polymeric nanoparticles. Antigen-encapsulated biodegradable nanoparticles have considerable potential for use in vaccine delivery systems. Although it is accepted that particle size is important for the induction of antigen-specific immune responses in vivo, little is known about how their size affects their intracellular fate. Here, we demonstrate that the size effects on the cellular uptake, intracellular degradation and distribution of protein-encapsulated nanoparticles. We prepared size-regulated ovalbumin (OVA)-encapsulated nanoparticles composed of hydrophobically modified poly(γ-glutamic acid) (γ-PGA). These nanoparticles were efficiently taken up by macrophages, and also delivered encapsulated OVA from the endosomes to the cytoplasm. Comparing 40-200 nm-sized nanoparticles, there was no significant difference in their intracellular distribution. Interestingly, the size of the nanoparticles affected the intracellular degradation of the encapsulated OVA. The uptake of OVA alone by macrophages resulted in early degradation of the OVA. In contrast, the degradation of OVA encapsulated into the nanoparticles was attenuated as compared to free OVA. A difference in OVA degradation kinetics was observed between the particle sizes, the degradation of small nanoparticles was slower than for the larger ones. These results indicate that particle size is an important factor for the intracellular degradation of encapsulated proteins and nanoparticles. These results will provide a rational design of nanoparticle-based vaccines to control immune responses.

Immunomodulatory nanoparticles as adjuvants and allergen-delivery system to human dendritic cells: Implications for specific immunotherapy.

Novel adjuvants and antigen-delivery systems with immunomodulatory properties that shift the allergenic Th2 response towards a Th1 or regulatory T cell response are desired for allergen-specific immunotherapy. This study demonstrates that 200-nm sized biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) are activators of human monocyte-derived dendritic cells (MoDCs). Gamma-PGA NPs are efficiently internalized by immature MoDCs and strongly stimulate production of chemokines and inflammatory cytokines as well as up-regulation of co-stimulatory molecules and immunomodulatory mediators involved in efficient T cell priming. Furthermore, MoDCs from allergic subjects stimulated in vitro with a mixture of gamma-PGA NPs and extract of grass pollen allergen Phleum pratense (Phl p) augment allergen-specific IL-10 production and proliferation of autologous CD4(+) memory T cells. Thus, gamma-PGA NPs are promising as sophisticated adjuvants and allergen-delivery systems in allergen-specific immunotherapy.

Induction of Potent CD8+ T-Cell Responses by Novel Biodegradable Nanoparticles Carrying Human Immunodeficiency Virus Type 1 gp120

ABSTRACT The mainstream of recent anti-AIDS vaccines is a prime/boost approach with multiple doses of the target DNA of human immunodeficiency virus type 1 (HIV-1) and recombinant viral vectors. In this study, we have attempted to construct an efficient protein-based vaccine using biodegradable poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs), which are capable of inducing potent cellular immunity. A significant expansion of CD8+ T cells specific to the major histocompatibility complex class I-restricted gp120 epitope was observed in mice intranasally immunized once with gp120-carrying NPs but not with gp120 alone or gp120 together with the B-subunit of cholera toxin. Both the gp120-encapsulating and -immobilizing forms of NPs could induce antigen-specific spleen CD8+ T cells having a functional profile of cytotoxic T lymphocytes. Long-lived memory CD8+ T cells could also be elicited. Although a substantial decay in the effector memory T cells was observed over time in the immunized mice, the central memory T cells remained relatively constant from day 30 to day 238 after immunization. Furthermore, the memory CD8+ T cells rapidly expanded with boosting with the same immunogen. In addition, γ-PGA NPs were found to be a much stronger inducer of antigen-specific CD8+ T-cell responses than nonbiodegradable polystyrene NPs. Thus, γ-PGA NPs carrying various HIV-1 antigens may have great potential as a novel priming and/or boosting tool in current vaccination regimens for the induction of cellular immune responses.

Formation of unimer nanoparticles by controlling the self-association of hydrophobically modified poly(amino acid)s.

Amphiphilic block or graft copolymers have been demonstrated to form a variety of self-assembled nano/microstructures in selective solvents. In this study, the self-association behavior of biodegradable graft copolymers composed of poly(γ-glutamic acid) (γ-PGA) as the hydrophilic segment and L-phenylalanine (Phe) as the hydrophobic segment in aqueous solution was investigated. The association behavior and unimer nanoparticle formation of these γ-PGA-graft-Phe (γ-PGA-Phe) copolymers in aqueous solution were characterized with a focus on the effect of the Phe grafting degree on the intra- and interpolymer association of γ-PGA-Phe. The particle size and number of polymer aggregates (N(agg)) in one particle of the γ-PGA-Phe depended on the Phe grafting degree. The size of γ-PGA-Phe with 12, 27, 35, or 42% Phe grafting (γ-PGA-Phe-12, -27, -35, or -42) was about 8-14 nm and the N(agg) was about 1, supporting the presence of a unimolecular graft copolymer in PBS. The pyrene fluorescence data indicated that γ-PGA-Phe-35 and -42 have hydrophobic domains formed by the intrapolymer association of Phe attached to γ-PGA. These results suggest that the Phe grafting degree is critical to the association behavior of γ-PGA-Phe and that γ-PGA-Phe-35 and -42 could form unimer nanoparticles. Moreover, when γ-PGA-Phe-42 dissolved in DMSO was added to various concentrations of NaCl solution, the particle size and N(agg) could be easily controlled by changing the NaCl concentration during the formation of the particles. These results suggest that biodegradable γ-PGA-Phe is useful for the fabrication of very small nanoparticles. It is expected that γ-PGA-Phe nanoparticles, including unimer particles, will have great potential as multifunctional carriers for pharmaceutical and biomedical applications, such as drug and vaccine delivery systems.