University of the Ryukyus · Tropical Biosphere Research Center · 教授

Research keywordstoxin・感染症ワクチン研究開発（獣医畜産、水産、医学）・Shiga・Protein

Research fieldsBacteriology (including Mycology)・Medicine・Medicine, Dentistry, and Pharmacy・Basic medicine・Experimental pathology・寄生虫学(含医用動物学)・Parasitology (including Sanitary zoology)・Applied veterinary science

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• B subunit of the type 2 Shiga toxin e variant (Stx2e) bundled by a five-stranded α-helical coiled coil protects piglets from porcine edema disease (vol. 61, 127140, 2025)2025 · Arakawa, T; Uefuji, H; Tamaki, Y; Oogai, S; Arakawa, H
• B subunit of the type 2 Shiga toxin e variant (Stx2e) bundled by a five-stranded α-helical coiled coil protects piglets from porcine edema disease2025 · Arakawa, T; Uefuji, H; Tamaki, Y; Oogai, S; Arakawa, H
• Shiga toxin type 2 B subunit protects mice against toxin challenge when leashed and bundled by a stable pentameric coiled-coil molecule2024 · Vaccines against Shiga toxin (Stx)-producing Escherichia coli (STEC) have not yet been developed. Two immunologically distinct serotypes of Stx (Stx1 and Stx2) are the main virulence factors of STEC. Thus, blocking their B subunits (StxB) from binding to the cell surface receptor globotriaosylceramide (Gb3) efficiently prevents the action of these toxins. We expressed Stx1B and Stx2B in E. coli inclusion bodies and reassembled them into pentamers by a stepwise dialysis. Stx1B pentamer fully protected mice against Stx1 challenge, but Stx2B pentamer failed to protect mice against Stx2 challenge. To explain those observations, we proposed that the pentamer of Stx2B readily dissociates into its constituent monomers, especially under in vivo conditions, thus being unable to induce pentamer-specific immunity. To increase pentamer stability, we fused the B subunit to a pentameric coiled-coil domain of the cartilage oligomeric matrix protein (COMP). This "five-to-five" fusion hybrid molecule (Stx2B-COMP) was shown to be protective against Stx2 challenge, demonstrating that the Stx2B subunit when leashed and bundled by a rigid pentameric coiled-coil domain mount a pentamer-specific immune response and efficiently neutralize the toxin both in vitro and in vivo. Our data strongly suggest that the Stx2B subunit moiety fluctuates between a pentameric and monomeric state within the fusion protein, which may increase the likelihood of the immune system recognizing the pentameric conformation for toxin neutralization.
• Shiga toxin type 2 B subunit protects mice against toxin challenge when leashed and bundled by a stable pentameric coiled-coil molecule.2024 · Vaccines against Shiga toxin (Stx)-producing Escherichia coli (STEC) have not yet been developed. Two immunologically distinct serotypes of Stx (Stx1 and Stx2) are the main virulence factors of STEC. Thus, blocking their B subunits (StxB) from binding to the cell surface receptor globotriaosylceramide (Gb3) efficiently prevents the action of these toxins. We expressed Stx1B and Stx2B in E. coli inclusion bodies and reassembled them into pentamers by a stepwise dialysis. Stx1B pentamer fully protected mice against Stx1 challenge, but Stx2B pentamer failed to protect mice against Stx2 challenge. To explain those observations, we proposed that the pentamer of Stx2B readily dissociates into its constituent monomers, especially under in vivo conditions, thus being unable to induce pentamer-specific immunity. To increase pentamer stability, we fused the B subunit to a pentameric coiled-coil domain of the cartilage oligomeric matrix protein (COMP). This “five-to-five” fusion hybrid molecule (Stx2B–COMP) was shown to be protective against Stx2 challenge, demonstrating that the Stx2B subunit when leashed and bundled by a rigid pentameric coiled-coil domain mount a pentamer-specific immune response and efficiently neutralize the toxin both in vitro and in vivo. Our data strongly suggest that the Stx2B subunit moiety fluctuates between a pentameric and monomeric state within the fusion protein, which may increase the likelihood of the immune system recognizing the pentameric conformation for toxin neutralization.

• VLP構成タンパク質の比率を制御するための技術基盤構築と組換えワクチン開発への応用2024 · 基盤研究(C)
• ２型志賀毒素B鎖５量体不安定性要因の分子機構解明と第２世代志賀毒素ワクチン開発2021 · 基盤研究(B)
• T=3正二十面体ウイルス粒子構造を模した分子構築法の開発と感染症ワクチンへの応用1925 · 本研究の目的は、本来3量体構造を取るタンパク質やドメイン構造を粒子状に再構築する技術の確立である。特にウイルス表層抗原には3量体構造を取るものが多く、それらを粒子形成させることで、その免疫原性は飛躍的に向上することが期待できる。この目的を達成するため、三角形分割数 T=3 正二十面体構造を有し、かつshell domain（S ドメイン）と protrusion domain（P ドメイン）が短い柔軟性の高いリンカーで繋がれたベータノダウイルス属に属する神経壊死性ウイルス (Nervous necrosis virus: NNV)の Sドメインを3量体抗原の“分子足場 (Molecular scaffold)”とし、任意の異種抗原を搭載した状態で粒子形成させる技術を確立する。 特にSドメインに3量体形成コイルドコイル分子 (CMP) を融合させた場合、NNV様粒子を形成することが示された。一方、Sドメイン単独では粒子形成しないことも分かった。よって、この結果はPドメインや他の3量体形成ドメインがSドメインの3量体形成の核として機能し、その後、粒子形成することを示唆している。さらにこの結果は、Pドメインを任意の3量体形成ドメインと置き換えても粒子形成可能なことを示している。以上の結果は、Scaffold 法が3量体構造をもつ異種抗原に対し、適応できる可能性を示唆しているといえる。
• 肺結核に対する感染防御CD8T細胞の誘導・活性化機構の解明とワクチンへの応用

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