Tomofumi Uto

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.

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.

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.

Modulation of innate and adaptive immunity by biodegradable nanoparticles.

Vaccine strategy needs efficient adjuvants to induce potent antigen-specific immune responses by targeting antigens to antigen presenting cells followed by their functional maturation. In this study, biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) were examined for their immunological activities in mice. Like lipopolysaccharide, gamma-PGA NPs strongly activated spleen dendritic cells (DCs) and induced their cytokine production and costimulatory molecule expression through the nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways. The immunization of mice with ovalbumin-carrying gamma-PGA NPs could induce the antigen-specific and long-lived effector and central memory CD8(+) T cells as well as antibody responses. Thus, gamma-PGA NPs have great potential as an efficient antigen carrier and strong adjuvant to DCs.

Manipulating the antigen-specific immune response by the hydrophobicity of amphiphilic poly(γ-glutamic acid) nanoparticles

The new generation vaccines are safe but poorly immunogenic, and thus they require the use of adjuvants. However, conventional vaccine adjuvants fail to induce potent cellular immunity, and their toxicity and side-effects hinder the clinical use. Therefore, a vaccine adjuvant which is safe and can induce an antigen-specific cellular immunity-biased immune response is urgently required. In the development of nanoparticle-based vaccine adjuvants, the hydrophobicity is one of the most important factors. It could control the interaction between the encapsulated antigens and/or nanoparticles with immune cells. In this study, nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid)-graft-L-phenylalanine ethyl ester (γ-PGA-Phe) with various grafting degrees of hydrophobic side chains were prepared to evaluate the effect of hydrophobicity of vaccine carriers on the antigen encapsulation behavior, cellular uptake, activation of dendritic cells (DCs), and induction of antigen-specific cellular immunity-biased immune responses. These NPs could efficiently encapsulate antigens, and the uptake amount of the encapsulated antigen by DCs was dependent on the hydrophobicity of γ-PGA-Phe NPs. Moreover, the activation potential of the DCs and the induction of antigen-specific cellular immunity were correlated with the hydrophobicity of γ-PGA-Phe NPs. By controlling the hydrophobicity of antigen-encapsulated γ-PGA-Phe NPs, the activation potential of DCs was able to manipulate about 5 to 30-hold than the conventional vaccine, and the cellular immunity was about 10 to 40-hold. These results suggest that the hydrophobicity of NPs is a key factor for changing the interaction between NPs and immune cells, and thus the induction of cellular immunity-biased immune response could be achieved by controlling the hydrophobicity of them.

Size effect of amphiphilic poly(γ-glutamic acid) nanoparticles on cellular uptake and maturation of dendritic cells in vivo.

We prepared size-regulated nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid) (γ-PGA). In this study, 40, 100 and 200 nm γ-PGA-graft-l-phenylalanine ethylester (γ-PGA-Phe) NPs were employed. The size of NPs significantly influenced the uptake and activation behaviors of antigen-presenting cells (APCs). When 40 nm γ-PGA-Phe NPs were applied to these cells in vitro, they were highly activated compared with 100 and 200 nm NPs, while cellular uptake was size dependent. The size of the γ-PGA-Phe NPs also significantly affected their migration to the lymph nodes and uptake behavior of NPs by dendritic cells (DCs) in vivo. The 40 nm γ-PGA-Phe NPs migrated more rapidly to the lymph nodes and were taken up by a greater number of DCs compared with 100 and 200 nm NPs. On the other hand, when the amount of γ-PGA-Phe NPs taken up per DC was evaluated, it was higher for 100 and 200 nm NPs than for 40 nm NPs, which suggests that the larger γ-PGA-Phe NPs can deliver a large amount of antigen to a single DC compared with smaller NPs. Furthermore, when examined the maturation of DCs in lymph nodes, 40 nm γ-PGA-Phe NPs efficiently stimulated DCs. These results suggest that the activation, uptake behavior by APCs, migration to lymph nodes, and DC maturation can be controlled by the size of γ-PGA-Phe NPs.

Synergistic stimulation of antigen presenting cells via TLR by combining CpG ODN and poly(γ-glutamic acid)-based nanoparticles as vaccine adjuvants.

CpG oligodeoxynucleotide (ODN) encapsulated poly(γ-glutamic acid)-graft-l-phenylalanine ethyl ester (γ-PGA-Phe) nanoparticles (NPs) employing polycations were prepared to develop vaccine delivery and adjuvant systems. The CpG ODN was stably encapsulated into the NPs when protamine was used as the polycation. The CpG ODN-encapsulated γ-PGA-Phe NPs were taken up by macrophages and CpG ODN which was encapsulated into the NPs internalized into endo/lysosomes, where the toll-like receptor (TLR) 9, which recognizes CpG ODN, is expressed. The examination of release behavior in vitro revealed that the encapsulated CpG ODN into NPs was released when these NPs were immersed into the early endosomal environment. Interestingly, CpG ODN-encapsulated γ-PGA-Phe NPs synergistically activated macrophages. This may be due to the multiple stimulation of TLRs by γ-PGA-Phe NPs (TLR4 ligand) and CpG ODN (TLR9 ligand). We previously reported that γ-PGA-Phe NPs are excellent vaccine adjuvants for inducing potent innate and adaptive immune responses via TLR4. Moreover, coencapsulated CpG ODN and antigen in γ-PGA-Phe NPs induced potent antigen-specific cellular immunity at a higher level than the mixture of CpG ODN and antigen which is the conventional vaccine system. These findings suggest that the conjugation strategies of biologically derived adjuvant and polymeric NPs will aid the development of a novel approach for safe and effective vaccine delivery and adjuvant systems.

Modulation of Gene Expression Related to Toll-Like Receptor Signaling in Dendritic Cells by Poly(γ-Glutamic Acid) Nanoparticles

ABSTRACT Poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) have previously been reported as an efficient antigen delivery system with adjuvant activity. In this study, the gene expression in murine bone marrow-derived dendritic cells (DCs) treated with γ-PGA NPs was examined by oligonucleotide microarray analysis and compared with that in cells treated with other adjuvants. The gene expression of proinflammatory chemokines, cytokines, and costimulatory molecules was upregulated considerably in DCs treated with γ-PGA NPs. The upregulation pattern was similar to that in DCs treated with lipopolysaccharide (LPS) but not to that in DCs treated with unparticulate γ-PGA. The activation of DCs by γ-PGA NPs was confirmed by real-time reverse transcriptase PCR (RT-PCR) analysis of genes related to Toll-like receptor (TLR) signaling. The effect of γ-PGA NPs on DCs was not annihilated by treatment with polymyxin B, an inhibitor of LPS. Furthermore, the immunization of mice with γ-PGA NPs carrying ovalbumin (OVA) as an antigen significantly induced antigen-specific CD8+ T cells and antigen-specific production of interleukin-2, tumor necrosis factor alpha, and gamma interferon from the cells. Such activities of γ-PGA NPs were more potent than those obtained with immunization with OVA plus aluminum hydroxide or OVA plus complete Freunds adjuvant. These results suggest that γ-PGA NPs induce a CD8+ T-cell response by activating innate immunity in a fashion different from that of LPS. Thus, γ-PGA NPs may be an attractive candidate to be developed further as a vaccine adjuvant.

Improvement of adaptive immunity by antigen-carrying biodegradable nanoparticles.

One of the most important aspects in vaccine development is to induce potent antigen-specific immune responses. In this study, we examined the immunological activities of antigen-carrying biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) in mice. The immunization with ovalbumin (OVA)-carrying gamma-PGA NPs (OVA-NPs) could induce significant expansion of antigen-specific CD8(+) T cells. Unlike complete Freunds adjuvant, subcutaneous (s.c.) inoculation of OVA-NPs to footpad did not generate injection site swelling. Although OVA-NPs could induce both antigen-specific cellular and humoral immune responses, the dominant induction of either cellular or humoral immunity was found to depend on their administration routes. Strong antibody production was observed by s.c. immunization, yet no antibody was identified by intranasal immunization. Thus, gamma-PGA NPs are a safe and efficient antigen carrier with unique immunological properties.

Plasmacytoid dendritic cells orchestrate TLR7-mediated innate and adaptive immunity for the initiation of autoimmune inflammation

Endosomal toll-like receptor (TLR)-mediated detection of viral nucleic acids (NAs) and production of type I interferon (IFN-I) are key elements of antiviral defense, while inappropriate recognition of self NAs with the induction of IFN-I responses is linked to autoimmunity such as psoriasis and systemic lupus erythematosus. Plasmacytoid dendritic cells (pDCs) are cells specialized in robust IFN-I secretion by the engagement of endosomal TLRs, and predominantly express sialic acid-binding Ig-like lectin (Siglec)-H. However, how pDCs control endosomal TLR-mediated immune responses that cause autoimmunity remains unclear. Here we show a critical role of pDCs in TLR7-mediated autoimmunity using gene-modified mice with impaired expression of Siglec-H and selective ablation of pDCs. pDCs were shown to be indispensable for the induction of systemic inflammation and effector T-cell responses triggered by TLR7 ligand. pDCs aggravated psoriasiform dermatitis mediated through the hyperproliferation of keratinocytes and enhanced dermal infiltration of granulocytes and γδ T cells. Furthermore, pDCs promoted the production of anti-self NA antibodies and glomerulonephritis in lupus-like disease by activating inflammatory monocytes. On the other hand, Siglec-H regulated the TLR7-mediated activation of pDCs. Thus, our findings reveal that pDCs provide an essential link between TLR7-mediated innate and adaptive immunity for the initiation of IFN-I-associated autoimmune inflammation.