Seishiro Naito

Antigen-loaded dissolving microneedle array as a novel tool for percutaneous vaccination.

Antigen-loaded dissolving microneedle array (dMNA) patches were investigated as novel systems for vaccine delivery into the skin, where immuno-competent dendritic cells are densely distributed. We fabricated micron-scale needles arrayed on patches, using chondroitin sulfate mixed with a model antigen, ovalbumin. Insertion of dMNA effectively delivered substantial amounts of ovalbumin into the skin within 3 min and induced robust antigen-specific antibody responses in the sera of mice. The antibody dose-response relationship showed that the efficiency of dMNA patch immunization was comparable to that of conventional intradermal injections. Thus, Antigen-loaded dMNA patches are a promising antigen-delivery system for percutaneous vaccination.

Application of DNA microarray technology to influenza A/Vietnam/1194/2004 (H5N1) vaccine safety evaluation

We propose that DNA microarray analysis can be used in the quality control of pandemic and endemic influenza vaccine. Based on the expression profiles of 76 genes in the rat lung one day after inoculation of influenza vaccine, we can distinguish whole-virion influenza vaccine (PDv: pandemic influenza vaccine and WPv: whole virion-particle vaccine) and sub-virion vaccine (HA vaccine) from saline. Among these 76 genes, we found genes up-regulated by influenza infection, as well as genes involved in the immune response, and interferon. Hierarchical clustering of each influenza vaccine by the expression profiles of these 76 genes matched data from current quality control tests in Japan, such as the abnormal toxicity test (ATT) and the leukopenic toxicity test (LTT). Thus, it can be concluded that DNA microarray technology is an informative, rapid and highly sensitive method with which to evaluate the quality of influenza vaccines. Using DNA microarray system, consistent with the results of the ATT and LTT, it was clarified that there was no difference in vaccine quality between PDv and WPv.

Ovalbumin-Liposome Conjugate Induces IgG but Not IgE Antibody Production

Antibody response after immunization with surface-coupled ovalbumin (OVA) of liposomes was investigated in mice. OVA was coupled to the surface of liposome via amino groups using glutaraldehyde. OVA-liposome conjugate induced a significant anti-OVA IgG antibody production in mice. However, no IgE antibody production specific for OVA was observed. Immunization with OVA-liposome induced IgE-specific unresponsiveness even after the subsequent challenge with OVA adsorbed with with aluminium hydroxide (OVA-alum), which induces a high level of IgE antibody production. Furthermore, following the primary immunization with OVA-alum, a secondary challenge with OVA-liposome boosted anti-OVA IgG but not anti-OVA IgE antibody production. These results show the potential of the antigen-liposome conjugate for the development of a vaccine with the least allergic reaction and also for the application of immunotherapy.

T Cell-Independent Regulation of IgE Antibody Production Induced by Surface-Linked Liposomal Antigen

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.

Epicutaneous immune modulation with Bet v 1 plus R848 suppresses allergic asthma in a murine model

Combining allergen(s) with an adjuvant is a strategy to improve the efficacy and safety of allergen‐specific immunotherapy. Here, we aimed at investigating the adjuvant effects of polyadenylic–polyuridylic acid (poly(A:U)), a TLR3 agonist, and R848 (resiquimod), a TLR7 agonist, in epicutaneous immunotherapy with Bet v 1, the major birch pollen allergen, to intervene in birch pollen allergy.

Antigen–Specific, IgE–Selective Unresponsiveness Induced by Antigen–Liposome Conjugates

Background: We have previously reported that ovalbumin (OVA) coupled with liposome via glutaraldehyde (GA) induced OVA–specific– and IgE–selective unresponsiveness in mice. Methods: In this study, OVA–liposome conjugates were made using four different coupling protocols: via GA, N–(6–maleimidocaproyloxy) succinimide (EMCS), disuccinimidyl suberate (DSS) and N–succimidyl–3(2–pyridyldithio)propionate (SPDP) and the induction of antigen–specific IgG and IgE antibody production was investigated for each. In addition, antigen–specific cytokine production by spleen cells of mice immunized either with OVA–liposome or with OVA adsorbed with aluminum hydroxide was investigated. Results: OVA–liposome conjugates coupled via GA or DSS did not induce anti–OVA IgE antibody production but induced substantial anti–OVA IgG antibody production. On the other hand, the induction of anti–OVA IgE unresponsiveness by OVA–liposome conjugates coupled via EMCS or SPDP was incomplete. The amount of interleukin 4 (IL–4) produced by spleen cells stimulated in vitro with OVA correlated well with anti–OVA IgE antibody production in donor mice. However, the production of no other cytokine, i.e., IL–2, IL–5, IL–10 or interferon–γ, was correlated with in vivo IgE antibody production. Conclusion: OVA–liposome coupled via GA or DSS induced complete suppression of anti–OVA IgE production. The results in this study further suggest that the regulation of IgE antibody production does not neccessarily correlate with so–called Th1 cytokine production.

Interferon regulatory factor‐2 induces megakaryopoiesis in mouse bone marrow hematopoietic cells

Megakaryopoiesis is associated with inflammatory reactions. To investigate the role of interferon regulatory factors (IRFs) in inflammation‐associated megakaryopoiesis, mouse bone marrow hematopoietic stem cells (HSCs) were analyzed. IFN‐γ treatment induced IRF‐2 expression as well as the expression of CD41 and IRF‐1 in HSCs. An in vitro clonogenic assay showed that IRF‐2‐ but not IRF‐1‐overexpressing cells increased the number of megakaryocytic colonies. IRF‐2 transfection up‐regulated CD41 promoter activity in hematopoietic cell lines. The number of CD41‐positive bone marrow cells increased in mice injected with IRF‐2‐expressing bone marrow cells. These findings suggest that IRF‐2 plays an important role in megakaryopoiesis in inflammatory states.

Protection of Monkeys against Shiga Toxin Induced by Shiga Toxin-Liposome Conjugates

Background: We previously reported that the purified Shiga toxins (Stx) Stx1 and Stx2, when coupled with liposomes, induced substantial production of anti-Stx1 and anti-Stx2 IgG antibody, respectively, in mice. The levels of anti-Stx antibody in the sera of mice immune to Stx-liposome correlated well with the protection against subsequent challenge with Stx. Furthermore, mice immunized with a mixture of Stx1-liposome and Stx2-liposome were successfully protected against oral infection with cytotoxin-producing Escherichia coli O157:H7. Methods: In this study, the induction of protection against Stx2 by Stx2-liposomes was evaluated in monkeys. Results: Stx2-liposomes induced a substantial amount of anti-Stx2 IgG antibodies as well as Stx2 neutralizing antibodies in monkeys. Test monkeys were successfully protected against challenge with lethal doses of Stx2. Moreover, these monkeys showed no apparent symptoms, while nonimmunized control monkeys died within 4 days with hemorrhagic gastroenteritis and renal disorder. In addition, as shown by other cases involving antigen-liposome conjugates, Stx2-liposome did not induce anti-Stx2 IgE antibody production, though it stimulated the production of a substantial amount of anti-Stx2 IgG antibodies. Conclusion: These results suggest that Stx-liposome conjugates may serve as candidate vaccines to induce protection against death caused by cytotoxin-producing E. coli infection.

Induction of Protection against Oral Infection with Cytotoxin–Producing Escherichia coli O157:H7 in Mice by Shiga–like Toxin–Liposome Conjugate

We have previously reported that purified Shiga–like toxins (SLT), SLT–I and SLT–II coupled with liposomes induced a substantial amount of anti–SLT–I and anti–SLT–II IgG antibody production, respectively, in mice. The levels of anti–SLT antibody in the sera of SLT–liposome–immune mice correlated well with the protection against subsequent challenge with SLT. In this study, mice were immunized intraperitoneally with the mixture of SLT–I–liposome and SLT–II–liposome and protection against oral infection with cytotoxin–producing Escherichia coli O157:H7 was evaluated. All of the mice that received immunization with the mixture of SLT–I–liposome and SLT–II–liposome were protected against subsequent intravenous challenge with 10 LD50 of either SLT–I or SLT–II. Eight weeks after primary immunization, mice were inoculated intragastrically with 109 CFU of E. coli O157:H7 strain 96–60. All SLT–liposome–immune mice tested survived without any apparent symptom while control mice died within 5 days. In addition, as shown by other antigen–liposome conjugates, SLT–liposome induced undetectable anti–SLT IgE antibody production while they induced substantial amounts of anti–SLT IgG antibodies. These results suggest that SLT–liposome conjugate may serve as a candidate vaccine that induces protection against cytotoxin–producing E. coli infection.

Application of quantitative gene expression analysis for pertussis vaccine safety control.

Although vaccines are routinely used to prevent infectious diseases, little is known about the comprehensive influences caused by vaccines. In this study, we showed, using comprehensive gene expression analysis, that pertussis vaccine affected many genes in multiple organs of vaccine-treated animals. In particular, lung was revealed to be the most suitable target to evaluate pertussis vaccine toxicity. The 13 genes identified from the analysis of vaccine-treated lung at day 1 showed a clear dendrogram corresponding to pertussis vaccine toxicity. Furthermore, quantitative analysis of these genes revealed a positive correlation between their respective expression levels and the degree of toxic effects observed in samples that had been treated with various doses of reference pertussis vaccines. The quantification of this 13 gene-set is an indicator of the vaccine toxicity-related reaction.