Effects of the Chemical Structures of Oligoarginines Conjugated to Biocompatible Polymers as a Mucosal Adjuvant on Antibody Induction in Nasal Cavities.

We have been investigating the potential of oligoarginine-linked polymers as an adjuvant for mucosal vaccination that induces immunoglobulin G (IgG) in systemic circulation and immunoglobulin A (IgA) secreted on the mucosa. Our latest infection experiments demonstrated that mice immunized nasally with a mixture of inactivated influenza viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) modified with D-octaarginine were perfectly protected from homologous virus infection. On the contrary, virus infection was observed in mice immunized with the antigen alone. This difference was presumably due to insignificant induction of secreted IgA on the nasal mucosa in the latter mice. Since it was unclear whether the current induction level was sufficient for heterologous virus infection, we evaluated the effects of the chemical structures of oligoarginines conjugated to PNVA-co-AA on induction of intranasal IgA. The number and optical activity of the arginine residues and the degree of modification with oligoarginines in the polymer backbone were listed as a factor that would influence IgA induction. Mouse experiments revealed that maximization of the modification resulted in an increase in adjuvant activities of oligoarginine-linked polymers most effectively. Glycine segments inserted between oligoarginines and the polymer backbone were a prerequisite for the maximization. The highest IgA level was observed when antigens were coadministered with diglycine-D-octaarginine-linked PNVA-co-AA.

Demonstration of d-Octaarginine-Linked Polymers as Promising Adjuvants for Mucosal Vaccination through Influenza Virus Challenge.

Mucosal vaccination is one of the most effective ways to reduce the risk of pandemics as a result of incorrect prediction of epidemic strains of influenza viruses or virus mutation. However, adjuvants and antigen carriers with potent immunostimulatory activities are a prerequisite for significant induction of mucosal immunity because most antigens are poorly immunogenic when solely applied to the mucosa. Our previous studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing d-octaarginine induced the secretion of antigen-specific immunoglobulin A (IgA) on the mucosa when nasally administered with virus antigens and that intranasal IgA reacts to viral strains other than the one used for immunization. Therefore, the present study evaluated capabilities of secreted IgA for protection against virus infection. When mice were inoculated with a mixture of inactivated H1N1 A/Puerto Rico/8/34 influenza viruses and d-octaarginine-linked polymers, antigen-specific secreted IgA was induced on the nasal mucosa. Immunized mice were completely protected from virus infection of the inoculated strain. To the contrary, mice nasally inoculated with inactivated viruses alone were infected with the homologous viruses presumably because of insignificant induction of secreted IgA. Results demonstrated that our polymer would be a promising adjuvant for mucosal vaccination.

Cell-penetrating peptide-linked polymers as carriers for mucosal vaccine delivery.

We evaluated the potential of poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, as a carrier for mucosal vaccine delivery. Mice were nasally inoculated four times every seventh day with PBS containing ovalbumin with or without the d-octaarginine-linked polymer. The polymer enhanced the production of ovalbumin-specific immunoglobulin G (IgG) and secreted immunoglobulin A (IgA) in the serum and the nasal cavity, respectively. Ovalbumin internalized into nasal epithelial cells appeared to stimulate IgA production. Ovalbumin transferred to systemic circulation possibly enhanced IgG production. An equivalent dose of the cholera toxin B subunit (CTB), which was used as a positive control, was superior to the polymer in enhancing antibody production; however, dose escalation of the polymer overcame this disadvantage. A similar immunization profile was also observed when ovalbumin was replaced with influenza virus HA vaccines. The polymer induced a vaccine-specific immune response identical to that induced by CTB, irrespective of the antibody type, when its dose was 10 times that of CTB. Our cell-penetrating peptide-linked polymer is a potential candidate for antigen carriers that induce humoral immunity on the mucosal surface and in systemic circulation when nasally coadministered with antigens.

Mechanism on antigen delivery under mucosal vaccination using cell-penetrating peptides immobilized at multiple points on polymeric platforms.

We have developed an aggregate of D-octaarginine immobilized at multiple points on a co-polymer of N-vinylacetamide and acrylic acid. Previous studies revealed that immunoglobulin G and A were induced when mice were inoculated with influenza virus antigens under coadministration with the D-octaarginine-immobilized polymers as a mucosal vaccine adjuvant. Infection experiments demonstrated that mice vaccinated with a mixture of inactivated influenza viruses and the polymers were protected from infection with mouse-adapted infectious viruses. In the present study, we investigated the mechanism on antigen delivery under mucosal vaccination using the polymers. Two-hour retention of fluorescein-labeled ovalbumin (F-OVA) on the nasal mucosa was observed when applied with the polymers; nevertheless F-OVA was eliminated less than 10 min under polymer-free conditions. F-OVA mixed with the polymers was vigorously taken up into murine dendritic cells. Electrophoresis and dynamic light scattering analysis indicated that OVA interacted with the polymers. The uptake of F-OVA was hardly ever inhibited by the addition of an excess amount of intact OVA. The results suggested that viral antigens were accumulated on the mucosa and delivered into dendritic cells under basolateral membranes via dendrites extending to the mucosal surface and/or subsequent to their permeation through epithelial cells, when they were coadministered with D-octaarginine-immobilized polymers.

Protein direct delivery to dendritic cells using nanoparticles based on amphiphilic poly(amino acid) derivatives.

Induction of an adaptive immune response by vaccination is possible for a broad range of infectious diseases or cancers. Antigen-loaded polymeric nanoparticles have recently been shown to possess significant potential as vaccine delivery systems and adjuvants. Here we demonstrate the use of nanoparticles composed of amphiphilic poly(amino acid) derivatives as vaccine adjuvants. We prepared protein-loaded, biodegradable nanoparticles composed of hydrophobically modified poly(gamma-glutamic acid) (gamma-PGA). gamma-PGA hydrophobic derivatives (gamma-hPGA) formed 200 nm-sized nanoparticles in water. The protein-encapsulated gamma-hPGA nanoparticles were efficiently taken up by immature dendritic cells (iDCs). Interestingly, the nanoparticle uptake by iDCs induced DC maturation. The immunization with human immunodeficiency virus (HIV)-1 gp120-encapsulated nanoparticles strongly induced antigen-specific cellular immunity. These results suggest that antigen-loaded gamma-hPGA nanoparticles provide a novel delivery tool for vaccination against viral infections or tumors. This system has potential application as a universal delivery system for protein-based vaccines capable of inducing cytotoxic T lymphocyte (CTL).

[Development of vaccine adjuvants using polymeric nanoparticles and their potential applications for anti-HIV vaccine].

The development of a prophylactic/therapeutic HIV-1 vaccine based on recombinant proteins is needed for the control of the worldwide AIDS epidemic. Subunit protein and peptide vaccines are generally very safe, with well-defined components. However, these antigens are often poorly immunogenic, and thus require the use of adjuvants to induce adequate immunity. Particulate adjuvants (e.g. micro/nanoparticles, emulsions, ISCOMS, liposomes, virosomes, and virus-like particles) have been widely investigated as HIV-1 vaccine delivery systems. Antigen uptake by antigen-presenting cells (APC) is enhanced by the association of the antigens with polymeric micro/nanoparticles. The adjuvant effect of micro/nanoparticles appears to largely be a consequence of their uptake into APC. More importantly, particulate antigens have been shown to be more efficient than soluble antigens for the induction of immune responses. Over the past two decades, we have studied the synthesis and clinical applications of core-corona polymeric nanospheres composed of hydrophobic polystyrene and hydrophilic macromonomers. Core-corona type polymeric nanospheres have applications in various technological and biomedical fields, because their chemical structures and particle size can be easily controlled. In this study, we focused on the development of a HIV-1 vaccine using polymeric nanoparticles. We evaluated the immunization strategies for HIV-1-capturing core-corona type polystyrene nanospheres that would efficiently induce HIV-1-specific IgA responses in female mice and the macaque genital tract. Moreover, based on this research, we attempted to develop novel biodegradable nanoparticles composed of poly (gamma-glutamic acid) (gamma-PGA) for protein-based vaccine delivery. These HIV-1-capturing nanospheres and protein-loaded gamma-PGA nanoparticles have shown unique potential as vaccine carriers.

Cross-reactivity of immunoglobulin A secreted on the nasal mucosa in mice nasally inoculated with inactivated H1N1 influenza A viruses in the presence of D-octaarginine-linked polymers.

We evaluated cross-reactivity of immunoglobulin A (IgA) secreted on the nasal mucosa in mice that were nasally inoculated 4 times with a mixture of inactivated H1N1 influenza A viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) bearing d-octaarginine at 7-day intervals. Three viral strains (A/Puerto Rico/8/34, A/New Caledonia/20/99 IVR116, and A/Solomon Islands/03/2006) and D-octaarginine-linked polymers with different molecular weights were used as antigens and their carriers, respectively. Secretion of intranasal IgA was barely observed when the inactivated virus alone was administered. The polymer induced the production of intranasal IgA specific to the inoculated viruses, irrespective of the viral strain and molecular weight of the polymer. The respective antibodies cross-reacted to recombinant hemagglutinin proteins of not only the viral strain used for immunization but also other H1N1 strains, including A/Puerto Rico/8/34 strain whose hemagglutinin proteins are diverse from those of other strains. Mice with high reactivity of IgA to the inoculated viruses tended to acquire clear cross-reactivity to other viral strains. Notably, IgA induced by inactivated H1N1 A/New Caledonia/20/99 IVR116 strain with the strongest immunogenicity between 3 antigens in the presence of the polymer cross-reacted to recombinant hemagglutinin proteins of the A/Brisbane/10/2007 and A/Viet Nam/1194/2004 strains, which are categorized into H3N2 and H5N1, respectively. Our polymer is a potential candidate for an efficient antigen carrier that induces mucosal IgA having cross-reactivity to antigenically drifted variants, irrespective of the subtype of viral strains.

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.

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(gamma-glutamic acid) (gamma-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, gamma-PGA NPs were found to be a much stronger inducer of antigen-specific CD8+ T-cell responses than nonbiodegradable polystyrene NPs. Thus, gamma-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.

IgG responses to intranasal immunization with cholera-toxin-immobilized polymeric nanospheres in mice.

IgG responses to antigen-nanosphere hybrids were studied in mice. Cholera toxin (CT) was covalently immobilized onto the surface of polymeric nanospheres (NS) with a nanophase-separated structure consisting of a polystyrene core and a poly(methacrylic acid) graft corona. Reaction conditions favoring the dehydroxide condensation reaction of the amino group of the CT with the carboxyl group of NS effectively immobilized CT onto their surface. When CT-immobilized nanospheres (CT-NS) were suspended in aqueous solution and administrated to mice either intranasally or intramuscularly, serum IgG titers elevated with increasing time and reached a maximum level at 8 weeks after immunization. On the other hand, intranasal administration of CT alone induced an even higher serum IgG titer than that of CT-NS at 4 weeks. However, the titer gradually decreased thereafter. Thus, polymeric NS may be an effective substrate to covalently immobilize antigen on their surface, steadily inducing a high level of IgG production in response to the intranasal administration.

Induction of HIV-specific antibody response and protection against vaginal SHIV transmission by intranasal immunization with inactivated SHIV-capturing nanospheres in macaques.

We have previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and that intranasal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody response in mice. In this study, to evaluate the protective effect of immunization, each three macaques was intranasally immunized with Con A-NS or inactivated simian/human immunodeficiency virus KU-2-capturing nanospheres (SHIV-NS) and then intravaginally challenged with a pathogenic virus, SHIV KU-2. After a series of six immunizations, vaginal anti-HIV-1 gp120 IgA and IgG antibodies were detected in all SHIV-NS-immunized macaques. After intravaginal challenge, one of the three macaques in each of the Con A-NS- and SHIV-NS-immunized groups was infected. Plasma viral RNA load of infected macaque in SHIV-NS-immunized macaques was substantially less than that in unimmunized control macaque and reached below the detectable level. However, it could not be determined whether intranasal immunization with SHIV-NS is effective in giving complete protection against intravaginal challenge. To explore the effect of the SHIV-NS vaccine, the remaining non-infected macaques were rechallenged intravenously with SHIV KU-2. After intravenous challenge, all macaques became infected. However, SHIV-NS-immunized macaques had lower viral RNA loads and higher CD4(+) T cell counts than unimmunized control macaques. Plasma anti-HIV-1 gp120 IgA and IgG antibodies were induced more rapidly in the SHIV-NS-immunized macaques than in the controls. The rapid antibody responses having neutralizing activity might contribute to the clearance of the challenge virus. Thus, SHIV-NS-immunized macaques exhibited partial protection to vaginal and systemic challenges with SHIV KU-2.