研究者データベース

関屋 俊輝(セキヤ トシキ)
人獣共通感染症リサーチセンター 生物製剤研究開発室
助教

基本情報

所属

  • 人獣共通感染症リサーチセンター 生物製剤研究開発室

職名

  • 助教

学位

  • 博士(免疫学・微生物学)(メルボルン大学)

J-Global ID

研究分野

  • ライフサイエンス / 免疫学

研究活動情報

論文

  • Marios Koutsakos, Toshiki Sekiya, Brendon Y Chua, Thi Hoang Oanh Nguyen, Adam K Wheatley, Jennifer A Juno, Marumi Ohno, Naoki Nomura, Yuki Ohara, Tomohiro Nishimura, Masafumi Endo, Saori Suzuki, Hirohito Ishigaki, Misako Nakayama, Cong T Nguyen, Yasushi Itoh, Masashi Shingai, Kazumasa Ogasawara, Yoichiro Kino, Stephen J Kent, David C Jackson, Lorena E Brown, Hiroshi Kida, Katherine Kedzierska
    Immunology and cell biology 2020年08月01日 [査読有り][通常論文]
     
    Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8+ and CD4+ T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models.
  • Marumi Ohno, Toshiki Sekiya, Naoki Nomura, Taku Ji Daito, Masashi Shingai, Hiroshi Kida
    Scientific reports 10 1 10879 - 10879 2020年07月02日 [査読有り][通常論文]
     
    Although the severity of influenza virus infections has been associated with host energy metabolism, the related mechanisms have not yet been clarified. Here we examined the effects of influenza virus infection on host energy metabolism in mice. After infecting mice with intranasal applications of 500 plaque-forming units of A/Puerto Rico/8/34 (H1N1; PR8) virus, the serum levels of most intermediates in the tricarboxylic acid (TCA) cycle and related metabolic pathways were significantly reduced. These data suggest that substrate supply to the TCA cycle is reduced under these conditions, rather than specific metabolic reactions being inhibited. Then, we focused on glucose and fatty acid metabolism that supply substrates to the TCA cycle. Akt phosphorylation following insulin injections was attenuated in the livers of PR8 virus-infected mice. Furthermore, glucose tolerance tests revealed that the PR8 virus-infected mice showed higher blood glucose levels than the vehicle-inoculated control mice. These results suggest that influenza virus infection impairs insulin signaling, which regulates glucose uptake. However, increases in the hepatic expressions of fatty acid-metabolizing enzymes suggest that fatty acids accumulate in liver cells of infected mice. Collectively, our data indicate that influenza virus infection dysregulates host energy metabolism. This line of investigation provides novel insights into the pathogenesis of influenza.
  • Toshiki Sekiya, Edin J Mifsud, Marumi Ohno, Naoki Nomura, Mayumi Sasada, Daisuke Fujikura, Takuji Daito, Masashi Shingai, Yuki Ohara, Tomohiro Nishimura, Masafumi Endo, Ryotarou Mitsumata, Tomio Ikeda, Hironori Hatanaka, Hiroki Kitayama, Kenji Motokawa, Tomoyoshi Sobue, Saori Suzuki, Yasushi Itoh, Lorena E Brown, Kazumasa Ogasawara, Yoichiro Kino, Hiroshi Kida
    Vaccine 37 15 2158 - 2166 2019年04月03日 [査読有り][通常論文]
     
    In contrast to current ether- or detergent-disrupted "split" vaccines (SVs) for influenza, inactivated whole influenza virus particle vaccines (WPVs) retain the original virus structure and components and as such may confer similar immunity to natural infection. In a collaboration between academia and industry, the potential of WPV as a new seasonal influenza vaccine was investigated. Each of the four seasonal influenza vaccine manufacturers in Japan prepared WPVs and SVs from the same batches of purified influenza virus. Both mice and monkeys vaccinated with the WPVs exhibited superior immune responses to those vaccinated with the corresponding SVs. Vaccination with A/California/07/2009 (H1N1) WPV enabled mice to survive a lethal challenge dose of homologous virus whereas those vaccinated with SV succumbed to infection within 6 days. Furthermore, mice vaccinated with WPV induced substantial numbers of multifunctional CD8+ T cells, important for control of antigenically drifted influenza virus strains. In addition, cytokines and chemokines were detected at early time points in the sera of mice vaccinated with WPV but not in those animals vaccinated with SV. These results indicate that WPVs induce enhanced innate and adaptive immune responses compared to equivalent doses of SVs. Notably, WPV at one fifth of the dose of SV was able to induce potent immunity with limited production of IL-6, one of the pyrogenic cytokines. We thus propose that WPVs with balanced immunogenicity and safety may set a new global standard for seasonal influenza vaccines.
  • Toshiki Sekiya, Junya Yamagishi, John Henry V. Gray, Paul G. Whitney, Axel Martinelli, Weiguang Zeng, Chinn Yi Wong, Chihiro Sugimoto, David C. Jackson, Brendon Y. Chua
    BIOMATERIALS 137 61 - 72 2017年08月 [査読有り][通常論文]
     
    The lipopeptide R4Pam2Cys is an agonist for toll-like receptor-2 (TLR2), a key pathogen-associated molecular pattern receptor expressed on many antigen-presenting cells such as dendritic cells (DCs). Electrostatic association of R4Pam2Cys with soluble protein antigens significantly enhances their immunogenicity and there is evidence to suggest that reducing the size of suitably adjuvanted-antigen complexes in solution may further improve their immunostimulatory capabilities. In this study, we investigated how incorporation of polyethylene glycol (PEG) into R4Pam2Cys affects the size, activity and efficacy of formed antigen-lipopeptide complexes. The presence of PEG was shown to increase solubility with a concomitant reduction in the particle size of vaccine formulations that was dependent on the length of PEG used. When compared to non-PEGylated R4Pam2Cys, vaccination of animals with antigen-complexed PEGylated R4Pam2Cys resulted not only in improvements in antibody production but significantly higher antigen-specific CD8(+) T cell responses. Both lipopeptides exhibited similar in vitro capabilities to induce DC maturation, facilitate antigen uptake and presentation to T cells. Moreover, analyses of the transcriptomes obtained from DCs treated with either lipopeptide revealed a large number of commonly induced genes with similar transcript expression levels, suggesting that common signalling pathways and processes were engaged following activation by either lipopeptide. In vivo analysis however revealed that vaccination with antigen-complexed PEGylated R4Pam2Cys resulted in improved antigen presentation to T cells. These heightened responses were not attributed to prolonged antigen persistence but rather due to more rapid transportation of antigen from the injection site into the draining lymph nodes over a short period of time. Our results indicate that reducing the size of formed antigen-TLR2-agonist complexes by PEGylation does not compromise the activity of the agonist but in fact enhances its trafficking in vivo ultimately leading to improved humoral and cell-mediated immune responses. (C) 2017 Elsevier Ltd. All rights reserved.
  • Brendon Y. Chua, Chinn Yi Wong, Edin J. Mifsud, Kathryn M. Edenborough, Toshiki Sekiya, Amabel C. L. Tan, Francesca Mercuri, Steve Rockman, Weisan Chen, Stephen J. Turner, Peter C. Doherty, Anne Kelso, Lorena E. Brown, David C. Jackson
    MBIO 6 6 e01024 - 15 2015年11月 [査読有り][通常論文]
     
    The continual threat to global health posed by influenza has led to increased efforts to improve the effectiveness of influenza vaccines for use in epidemics and pandemics. We show in this study that formulation of a low dose of inactivated detergent-split influenza vaccine with a Toll-like receptor 2 (TLR2) agonist-based lipopeptide adjuvant (R(4)Pam(2)Cys) provides (i) immediate, antigen-independent immunity mediated by the innate immune system and (ii) significant enhancement of antigen-dependent immunity which exhibits an increased breadth of effector function. Intranasal administration of mice with vaccine formulated with R(4)Pam(2)Cys but not vaccine alone provides protection against both homologous and serologically distinct (heterologous) viral strains within a day of administration. Vaccination in the presence of R(4)Pam(2)Cys subsequently also induces high levels of systemic IgM, IgG1, and IgG2b antibodies and pulmonary IgA antibodies that inhibit hemagglutination (HA) and neuraminidase (NA) activities of homologous but not heterologous virus. Improved primary virus nucleoprotein (NP)-specific CD8(+) T cell responses are also induced by the use of R(4)Pam(2)Cys and are associated with robust recall responses to provide heterologous protection. These protective effects are demonstrated in wild-type and antibody-deficient animals but not in those depleted of CD8(+) T cells. Using a contact-dependent virus transmission model, we also found that heterologous virus transmission from vaccinated mice to naive mice is significantly reduced. These results demonstrate the potential of adding a TLR2 agonist to an existing seasonal influenza vaccine to improve its utility by inducing immediate short-term nonspecific antiviral protection and also antigen-specific responses to provide homologous and heterologous immunity. IMPORTANCE The innate and adaptive immune systems differ in mechanisms, specificities, and times at which they take effect. The innate immune system responds within hours of exposure to infectious agents, while adaptive immunity takes several days to become effective. Here we show, by using a simple lipopeptide-based TLR2 agonist, that an influenza detergent-split vaccine can be made to simultaneously stimulate and amplify both systems to provide immediate antiviral protection while giving the adaptive immune system time to implement long-term immunity. Both types of immunity induced by this approach protect against vaccine-matched as well as unrelated virus strains and potentially even against strains yet to be encountered. Conferring dual functionality to influenza vaccines is beneficial for improving community protection, particularly during periods between the onset of an outbreak and the time when a vaccine becomes available or in scenarios in which mass vaccination with a strain to which the population is immunologically naive is imperative.
  • Brendon Y. Chua, Toshiki Sekiya, Mohammad Al Kobaisi, Kirsty R. Short, David E. Mainwaring, David C. Jackson
    BIOMATERIALS 53 50 - 57 2015年06月 [査読有り][通常論文]
     
    In this study, we describe a biodegradable vaccine depot which persists in vivo for at least 4-months, provides synergistic adjuvant effects and also allows dose sparing of both antigen and adjuvant. A single administration results in immediate release of a priming dose of vaccine, by a process of syneresis, which is then followed by release of remaining vaccine which maintains robust antibody levels that last for more than a year. The platform technology comprises two aqueous components; one contains chitosan and hydroxyapatite, in which the vaccine is incorporated, and the other consists of a crosslinking agent, tripolyphosphate (TPP) and chondroitin sulphate. When co-injected into tissue, they spontaneously crosslink forming a firm yet compliant vaccine-containing depot. Whole body imaging of animals inoculated with the material show that the depot persists in situ for up to 19 weeks. Vaccination of mice with depot formulations containing ovalbumin (OVA) emulsified in Montanide ISA 61 adjuvant results in the induction of robust antibody responses using doses of adjuvant 40-fold less than those recommended by the manufacturer. Dose sparing effects were also apparent with antigen when delivered in the depot. Similar dose sparing effects were observed with Montanide ISA 50, complete and incomplete Freund's adjuvants but not with aluminium hydroxide nor Quil A. Antibody titres, induced by a single dose of antigen/adjuvant formulation incorporated in the depot, persisted at high levels for at least 55 weeks following a single dose of vaccine. (C) 2015 Elsevier Ltd. All rights reserved.
  • Brendon Y. Chua, Matthew R. Olson, Sammy Bedoui, Toshiki Sekiya, Chinn Y. Wong, Stephen J. Turner, David C. Jackson
    IMMUNOLOGY AND CELL BIOLOGY 92 4 377 - 383 2014年04月 [査読有り][通常論文]
     
    We have previously shown that the immunogenicity of protein antigens can be significantly enhanced if electrostatically associated with the Toll-like receptor-2 agonist-based lipopeptide R(4)Pam(2)Cys. The precise mechanisms and effectiveness of the cytotoxic T-lymphocyte (CTL)-mediated response facilitated by this agonist, however, have not been studied. Here we show that priming by dendritic cells (DCs) in the draining lymph nodes of animals vaccinated with antigen delivered using R(4)Pam(2)Cys results in significantly improved T-cell proliferation and induces their differentiation into polyfunctional effector CTLs characterised by granzyme B expression and the ability to secrete interferon-gamma, interleukin-2 and tumor necrosis factor-alpha 7 days after vaccination. After 30 days, frequencies of antigen-specific CD62(low)CD127(high) (effector memory), CD62(high)CD127(high) (central memory) and CD43(low)CD27(high) CD8(+) T cells, a phenotype associated with strong recall responses against respiratory infections, are also increased compared with responses obtained with antigens formulated in the adjuvants Alum (alhydrogel) and CFA (complete Freund's adjuvant). The phenotypic changes observed in these mice vaccinated using R(4)Pam(2)Cys further correlated with their ability to recall specific T cells into the lung to mediate the reduction of pulmonary viral titres following challenge with a chimeric influenza virus containing the K(b)OVA(257-264) epitope compared with animals vaccinated using Alum or CFA. The findings from this study not only demonstrate that better T-cell responses can be elicited using R(4)Pam(2)Cys compared with classically utilised adjuvants but also highlight the potential effectiveness of this lipopeptide-based adjuvant particularly against viral infections that require resolution through cell-mediated immunity.
  • Brendon Y. Chua, Douglas Johnson, Amabel Tan, Linda Earnest-Silveira, Toshiki Sekiya, Ruth Chin, Joseph Torresi, David C. Jackson
    PLOS ONE 7 10 e47492  2012年10月 [査読有り][通常論文]
     
    Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. In this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the TLR2 agonist Pam(2)Cys (E(8)Pam(2)Cys) to enhance the immunogenicity of VLPs containing the HCV structural proteins (core, E1 and E2) of genotype 1a. While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum). Dramatically improved VLP and E2-specific antibody responses were observed in VLP+E(8)Pam(2)Cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted VLPs was required to match the antibody titres obtained with a single dose of VLPs formulated with this lipopeptide. This result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of VLP+E(8)Pam(2)Cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of VLPs by Huh7 cells. Moreover, vaccination of HLA-A2 transgenic mice with this formulation also induced better VLP-specific IFN-gamma-mediated responses compared to non-adjuvanted VLPs but comparable levels to that achieved when coadministered with complete freund's adjuvant. These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV.

その他活動・業績

  • Brendon Y Chua, Toshiki Sekiya, David C Jackson Viral immunology 31 (2) 150 -158 2018年03月 [査読有り][通常論文]
     
    Empirically derived vaccines have in the past relied on the isolation and growth of disease-causing microorganisms that are then inactivated or attenuated before being administered. This is often done without prior knowledge of the mechanisms involved in conferring protective immunity. Recent advances in scientific technologies and in our knowledge of how protective immune responses are induced enable us to rationally design novel and safer vaccination strategies. Such advances have accelerated the development of inactivated whole-organism- and subunit-based vaccines. In this review, we discuss ideal attributes and criteria that need to be considered for the development of vaccines and some existing vaccine platforms. We focus on inactivated vaccines against influenza virus and ways by which vaccine efficacy can be improved with the use of adjuvants and Toll-like receptor-2 signaling.


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