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Fukuhara Hideo

International Institute for Zoonosis Control　Division of Pathogen StructureAssociate Professor
Institute for Integrated Innovations　Institute for Vaccine Research and DevelopmentAssociate Professor

Researcher basic information

■ Degree
  • 博士（農学）, 九州大学
■ URL
researchmap URL■ Various IDs
J-Global ID■ Research Keywords and Fields
Research Field
  • Life Science, Structural biochemistry
■ Educational Organization

Research activity information

■ Papers
  • Understanding the structure of measles virus and its implications for novel drug discovery
    Liuan Chen; Shunsuke Kita; Hideo Fukuhara; Katsumi Maenaka
    Expert Opinion on Drug Discovery, 20, 9, 1131, 1140, Informa UK Limited, 18 Aug. 2025
    Scientific journal
  • ARNAX is an ideal adjuvant for COVID-19 vaccines to enhance antigen-specific CD4+ and CD8+ T-cell responses and neutralizing antibody induction.
    Tomomi Kawakita; Toshiki Sekiya; Yayoi Kameda; Naoki Nomura; Marumi Ohno; Chimuka Handabile; Akari Yamaya; Hideo Fukuhara; Yuki Anraku; Shunsuke Kita; Shinsuke Toba; Hirotake Tsukamoto; Tomohiro Sawa; Hiroyuki Oshiumi; Yasushi Itoh; Katsumi Maenaka; Akihiko Sato; Hirofumi Sawa; Yasuhiko Suzuki; Lorena E Brown; David C Jackson; Hiroshi Kida; Misako Matsumoto; Tsukasa Seya; Masashi Shingai
    Journal of virology, e0229024, 15 Apr. 2025, [International Magazine]
    English, Scientific journal, UNLABELLED: ARNAX is a synthetic nucleotide-based Toll-like receptor 3 (TLR3) ligand that specifically stimulates the TLR3/TIR domain-containing adaptor molecule 1 (TICAM-1) pathway without activating inflammatory responses. ARNAX activates cellular immunity via cross-presentation; hence, its practical application has been demonstrated in cancer immunotherapy. Given the importance of cellular immunity in virus infections, ARNAX is expected to be a more effective vaccine adjuvant for virus infections than alum, an adjuvant approved for human use that mainly enhances humoral immunity. In the present study, the trimeric recombinant spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was prepared as a vaccine antigen and formulated with ARNAX. When T-cell and neutralizing antibody responses were evaluated in immunized mice, antigen formulated with ARNAX generated significantly larger numbers of antigenspecific CD4+ and CD8+ T cells, as well as higher titers of neutralizing antibodies, compared to antigen alone or antigen formulated with alum. In experiments where immunized mice were challenged with a SARS-CoV-2 mouse-adapted virus derived from the ancestral strain, immunization with antigen formulated with ARNAX reduced virus titers in the lungs at 3 days post-infection to a much greater extent than did immunization with either antigen alone or that formulated with alum. These results show that ARNAX potently enhances the levels of both cellular and humoral immunity above those seen with alum, providing significantly greater viral clearing responses. Thus, ARNAX may act as a useful adjuvant for prophylactic vaccines, particularly for viral infectious diseases. IMPORTANCE: Cellular immunity is a critical immunological defense system against virus infections. However, aluminum salts, the most widely used adjuvant for vaccines for human use, do not promote strong cellular immunity. To prepare for the next pandemic of viral origin, the development of Th1-type adjuvants with low adverse reactions that induce cellular immunity is necessary. ARNAX is a TLR3 agonist consisting of DNA-RNA hybrid nucleic acid, which is expected to be an adjuvant that induces cellular immunity. The present study using a coronavirus disease 2019 mouse model demonstrated that ARNAX potently induces cellular immunity in addition to humoral immunity with minimal induction of inflammatory cytokines. Therefore, ARNAX has the potential to be used as a potent and welltolerated adjuvant for vaccines against pandemic viruses emerging in the future.
  • Structural and virological identification of neutralizing antibody footprint provides insights into therapeutic antibody design against SARS-CoV-2 variants.
    Yuki Anraku; Shunsuke Kita; Taishi Onodera; Akihiko Sato; Takashi Tadokoro; Shiori Ito; Yu Adachi; Ryutaro Kotaki; Tateki Suzuki; Jiei Sasaki; Nozomi Shiwa-Sudo; Naoko Iwata-Yoshikawa; Noriyo Nagata; Souta Kobayashi; Yasuhiro Kazuki; Mitsuo Oshimura; Takao Nomura; Michihito Sasaki; Yasuko Orba; Tadaki Suzuki; Hirofumi Sawa; Takao Hashiguchi; Hideo Fukuhara; Yoshimasa Takahashi; Katsumi Maenaka
    Communications biology, 8, 1, 483, 483, 22 Mar. 2025, [International Magazine]
    English, Scientific journal, Medical treatments using potent neutralizing SARS-CoV-2 antibodies have achieved remarkable improvements in clinical symptoms, changing the situation for the severity of COVID-19 patients. We previously reported an antibody, NT-108 with potent neutralizing activity. However, the structural and functional basis for the neutralizing activity of NT-108 has not yet been understood. Here, we demonstrated the therapeutic effects of NT-108 in a hamster model and its protective effects at low doses. Furthermore, we determined the cryo-EM structure of NT-108 in complex with SARS-CoV-2 spike. The single-chain Fv construction of NT-108 improved the cryo-EM maps because of the prevention of preferred orientations induced by Fab orientation. The footprints of NT-108 illuminated how escape mutations such as E484K evade from class 2 antibody recognition without ACE2 affinity attenuation. The functional and structural basis for the potent neutralizing activity of NT-108 provides insights into the rational design of therapeutic antibodies.
  • Evolutionary and structural basis of SLAM utilization in morbilliviruses – Its implications for host range and cross-species transmission
    Ayumu Hyodo; Fumio Seki; Kento Fukuda; Kaede Tashiro; Yuki Kitai; Yukiko Akahori; Hideko Watabe; Hiroshi Katoh; Rikuto Osaki; Daisuke Takaya; Norihito Kawashita; Hideo Fukuhara; Satoshi Ikegame; Tomoki Yoshikawa; Park Eunsil; Shigeru Morikawa; Ryoji Yamaguchi; Benhur Lee; Katsumi Maenaka; Tsuyoshi Shirai; Kaori Fukuzawa; Shigenori Tanaka; Makoto Takeda
    PLOS Pathogens, 21, 6, e1012990, e1012990, Cold Spring Harbor Laboratory, 19 Feb. 2025
    Scientific journal, Abstract

    Morbilliviruses, including measles virus (MV), canine distemper virus (CDV), and cetacean morbillivirus, pose a significant threat to humans and animals. While the host range of morbilliviruses is generally well-defined, severe cross-species transmission events also have been reported. Their entry into immune cells, the primary targets of morbilliviruses, relies on the signaling lymphocytic activation molecule (SLAM), a receptor whose species-specific variations influence viral host range. However, the extent to which SLAM diversity is a barrier to cross-species transmission remains poorly understood. In this study, we systematically investigated SLAM-mediated host specificity. We found that most morbilliviruses efficiently utilize SLAM from multiple host species, except for human SLAM. Among the morbilliviruses tested, only MV efficiently utilized human SLAM.

    Bats are natural reservoirs for many zoonotic viruses. Recent discoveries of novel morbilliviruses in bats provide new insights into morbillivirus evolution. Bats may have played a significant role in morbillivirus evolution because bat (Myotis) SLAM also functioned as an efficient receptor for multiple morbilliviruses. Unlike other morbilliviruses, MV utilizedMyotisbat SLAM inefficiently. We conducted an MV adaptation experiment withMyotisbat SLAM to better understand SLAM recognition by morbilliviruses. MV readily adapted to utilizeMyotisbat SLAM by acquiring a single N187Y mutation in its hemagglutinin protein, and computational structural modeling and fragment molecular orbital calculations with molecular dynamics simulations revealed key interaction changes that facilitated MV’s adaptation toMyotisbat SLAM. These findings highlight the adaptability of morbilliviruses in utilizing diverse animal SLAMs. Notably, hypothetical ancestral SLAMs reconstructed in this study acted as universal receptors for all morbilliviruses. These results reinforced that morbillivirus receptor usage is primarily supported by evolutionarily conserved structural features of SLAM, highlighting a molecular basis that enables morbilliviruses to rapidly adapt to diverse animal SLAMs.

    Author Summary

    Our study explores how viruses in the genus morbillivirus, such as measles virus, canine distemper virus, and rinderpest virus, which are notorious for deadly outbreaks in humans and animals, can jump between species. The host range of morbilliviruses is significantly influenced by a receptor molecule on cell surfaces known as the signaling lymphocytic activating molecule (SLAM). By examining SLAMs from various animals, including humans, dolphins, dogs, seals, and bats, we observed how these viruses can infect or adapt to infect different hosts. We found that in some cases slight differences in SLAM may act as initial barriers to cross-species transmission. However, these viruses rapidly overcome such barriers, showing a remarkable ability to adapt. Our research highlights the importance of monitoring these viruses to predict and prevent potential cross-species infections, which is crucial for protecting public health and animal welfare over time.
  • Glycan-shielded homodimer structure and dynamical features of the canine distemper virus hemagglutinin relevant for viral entry and efficient vaccination.
    Hideo Fukuhara; Kohei Yumoto; Miyuki Sako; Mizuho Kajikawa; Toyoyuki Ose; Mihiro Kawamura; Mei Yoda; Surui Chen; Yuri Ito; Shin Takeda; Mwila Mwaba; Jiaqi Wang; Takao Hashiguchi; Jun Kamishikiryo; Nobuo Maita; Chihiro Kitatsuji; Makoto Takeda; Kimiko Kuroki; Katsumi Maenaka
    eLife, 12, 24 Jul. 2024, [International Magazine]
    English, Scientific journal, Canine distemper virus (CDV) belongs to morbillivirus, including measles virus (MeV) and rinderpest virus, which causes serious immunological and neurological disorders in carnivores, including dogs and rhesus monkeys, as recently reported, but their vaccines are highly effective. The attachment glycoprotein hemagglutinin (CDV-H) at the CDV surface utilizes signaling lymphocyte activation molecule (SLAM) and Nectin-4 (also called poliovirus-receptor-like-4; PVRL4) as entry receptors. Although fusion models have been proposed, the molecular mechanism of morbillivirus fusion entry is poorly understood. Here, we determined the crystal structure of the globular head domain of CDV-H vaccine strain at 3.2 Å resolution, revealing that CDV-H exhibits a highly tilted homodimeric form with a six-bladed β-propeller fold. While the predicted Nectin-4-binding site is well conserved with that of MeV-H, that of SLAM is similar but partially different, which is expected to contribute to host specificity. Five N-linked sugars covered a broad area of the CDV-H surface to expose receptor-binding sites only, supporting the effective production of neutralizing antibodies. These features are common to MeV-H, although the glycosylation sites are completely different. Furthermore, real-time observation using high-speed atomic force microscopy revealed highly mobile features of the CDV-H dimeric head via the connector region. These results suggest that sugar-shielded tilted homodimeric structure and dynamic conformational changes are common characteristics of morbilliviruses and ensure effective fusion entry and vaccination.
  • 【あなたのラボから薬を生み出す アカデミア創薬の実践 All JAPAN体制の先端技術支援を利用した創薬の最前線】(第2章)創薬標的タンパク質の構造解析と分子設計の革新的進歩 BSL3クライオ電子顕微鏡を用いた感染症創薬・ワクチン研究とその展望
    前仲 勝実; 福原 秀雄; Dokainish Hisham; 安楽 佑樹; 喜多 俊介
    実験医学, 42, 2, 246, 252, (株)羊土社, Feb. 2024
    Japanese
  • Rational in silico design identifies two mutations that restore UT28K SARS-CoV-2 monoclonal antibody activity against Omicron BA.1
    Tatsuhiko Ozawa; Yoshiki Ikeda; Liuan Chen; Rigel Suzuki; Atsushi Hoshino; Akira Noguchi; Shunsuke Kita; Yuki Anraku; Emiko Igarashi; Yumiko Saga; Noriko Inasaki; Shunta Taminishi; Jiei Sasaki; Yuhei Kirita; Hideo Fukuhara; Katsumi Maenaka; Takao Hashiguchi; Takasuke Fukuhara; Kenichi Hirabayashi; Hideki Tani; Hiroyuki Kishi; Hideki Niimi
    Structure, Elsevier BV, Jan. 2024
    Scientific journal
  • Unprecedented spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions
    Hideo Fukuhara; Hisham M. Dokainish; Shunsuke Kita; Koshiro Tabata; Akira Takasu; Juha T. Huiskonen; Yuki Anraku; Toshiya Senda; David I. Stuart; Michihito Sasaki; Yasuko Orba; Yasuhiko Suzuki; Hirofumi Sawa; Katsumi Maenaka
    11 Oct. 2023
  • Structural delineation and computational design of SARS-CoV-2-neutralizing antibodies against Omicron subvariants.
    Saya Moriyama; Yuki Anraku; Shunta Taminishi; Yu Adachi; Daisuke Kuroda; Shunsuke Kita; Yusuke Higuchi; Yuhei Kirita; Ryutaro Kotaki; Keisuke Tonouchi; Kohei Yumoto; Tateki Suzuki; Taiyou Someya; Hideo Fukuhara; Yudai Kuroda; Tsukasa Yamamoto; Taishi Onodera; Shuetsu Fukushi; Ken Maeda; Fukumi Nakamura-Uchiyama; Takao Hashiguchi; Atsushi Hoshino; Katsumi Maenaka; Yoshimasa Takahashi
    Nature communications, 14, 1, 4198, 4198, 14 Jul. 2023, [International Magazine]
    English, Scientific journal, SARS-CoV-2 Omicron subvariants have evolved to evade receptor-binding site (RBS) antibodies that exist in diverse individuals as public antibody clones. We rationally selected RBS antibodies resilient to mutations in emerging Omicron subvariants. Y489 was identified as a site of virus vulnerability and a common footprint of broadly neutralizing antibodies against the subvariants. Multiple Y489-binding antibodies were encoded by public clonotypes and additionally recognized F486, potentially accounting for the emergence of Omicron subvariants harboring the F486V mutation. However, a subclass of antibodies broadly neutralized BA.4/BA.5 variants via hydrophobic binding sites of rare clonotypes along with high mutation-resilience under escape mutation screening. A computationally designed antibody based on one of the Y489-binding antibodies, NIV-10/FD03, was able to bind XBB with any 486 mutation and neutralized XBB.1.5. The structural basis for the mutation-resilience of this Y489-binding antibody group may provide important insights into the design of therapeutics resistant to viral escape.
  • Structural delineation of SARS-CoV-2 broadly neutralizing antibody to Omicron subvariants including BA.4/BA.5 and BA.2.75
    Saya Moriyama; Yuki Anraku; Shunta Taminishi; Yu Adachi; Daisuke Kuroda; Shunsuke Kita; Yusuke Higuchi; Ryutaro Kotaki; Keisuke Tonouchi; Kohei Yumoto; Tateki Suzuki; Taiyou Someya; Hideo Fukuhara; Yudai Kuroda; Tsukasa Yamamoto; Taishi Onodera; Shuetsu Fukushi; Ken Maeda; Fukumi Nakamura-Uchiyama; Takao Hashiguchi; Atsushi Hoshino; Katsumi Maenaka; Yoshimasa Takahashi
    Research Square Platform LLC, 21 Dec. 2022
    Abstract

    SARS-CoV-2 Omicron subvariants have evolved to evade receptor-binding site (RBS) antibodies that exist in diverse individuals as public antibody clones. We rationally selected RBS antibodies resilient to mutations in emerging Omicron subvariants. Y489 was identified as a site of virus vulnerability and a common footprint of broadly neutralizing antibodies against the subvariants. Multiple Y489-binding antibodies were encoded by public clonotypes and additionally recognized F486, potentially accounting for the emergence of Omicron subvariants harboring the F486V mutation. However, a subclass of antibodies broadly neutralized BA.4/BA.5 variants via hydrophobic binding sites of rare clonotypes along with extremely high mutation-resilience under escape mutation screening. The structural basis for mutation-resilience of this antibody group may inform the design of therapeutics resistant to viral escape.
  • Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant.
    Akatsuki Saito; Tomokazu Tamura; Jiri Zahradnik; Sayaka Deguchi; Koshiro Tabata; Yuki Anraku; Izumi Kimura; Jumpei Ito; Daichi Yamasoba; Hesham Nasser; Mako Toyoda; Kayoko Nagata; Keiya Uriu; Yusuke Kosugi; Shigeru Fujita; Maya Shofa; Mst Monira Begum; Ryo Shimizu; Yoshitaka Oda; Rigel Suzuki; Hayato Ito; Naganori Nao; Lei Wang; Masumi Tsuda; Kumiko Yoshimatsu; Jin Kuramochi; Shunsuke Kita; Kaori Sasaki-Tabata; Hideo Fukuhara; Katsumi Maenaka; Yuki Yamamoto; Tetsuharu Nagamoto; Hiroyuki Asakura; Mami Nagashima; Kenji Sadamasu; Kazuhisa Yoshimura; Takamasa Ueno; Gideon Schreiber; Akifumi Takaori-Kondo; Kotaro Shirakawa; Hirofumi Sawa; Takashi Irie; Takao Hashiguchi; Kazuo Takayama; Keita Matsuno; Shinya Tanaka; Terumasa Ikeda; Takasuke Fukuhara; Kei Sato
    Cell host & microbe, 30, 11, 1540, 1555, 09 Nov. 2022, [International Magazine]
    English, Scientific journal, The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency, but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.
  • 構造解析に向けたヒト免疫不全ウイルス2(HIV-2)エンベロープ糖タンパク質の調製(Preparation of human immunodeficiency virus type-2(HIV-2) envelope glycoprotein for structure analysis)
    Anraku Yuki; Kita Shunsuke; Fukuhara Hideo; Kawabata Haruka; Akiyama Takaki; Davis Simon; Furukawa Atsushi; de Silva Thushan I.; Robinson James E.; Zhao Yuguang; Jones E. Yvonne; Stuart David; Huiskonen Juha T.; Rowland-Jones Sarah; Maenaka Katsumi
    生物物理, 62, Suppl.1-2, S350, S350, (一社)日本生物物理学会, Aug. 2022
    English
  • Structure of the human galanin receptor 2 bound to galanin and Gq reveals the basis of ligand specificity and how binding affects the G-protein interface.
    Yunseok Heo; Naito Ishimoto; Ye-Eun Jeon; Ji-Hye Yun; Mio Ohki; Yuki Anraku; Mina Sasaki; Shunsuke Kita; Hideo Fukuhara; Tatsuya Ikuta; Kouki Kawakami; Asuka Inoue; Katsumi Maenaka; Jeremy R H Tame; Weontae Lee; Sam-Yong Park
    PLoS biology, 20, 8, e3001714, Aug. 2022, [International Magazine]
    English, Scientific journal, Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer's disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hβ2AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.
  • Novel super-neutralizing antibody UT28K is capable of protecting against infection from a wide variety of SARS-CoV-2 variants
    Tatsuhiko Ozawa; Hideki Tani; Yuki Anraku; Shunsuke Kita; Emiko Igarashi; Yumiko Saga; Noriko Inasaki; Hitoshi Kawasuji; Hiroshi Yamada; so Ichiro Sasaki; Mayu Somekawa; Jiei Sasaki; Yoshihiro Hayakawa; Yoshihiro Yamamoto; Yoshitomo Morinaga; Nobuyuki Kurosawa; Masaharu Isobe; Hideo Fukuhara; Katsumi Maenaka; Takao Hashiguchi; Hiroyuki Kishi; Isao Kitajima; Shigeru Saito; Hideki Niimi
    mAbs, 14, 1, e2072455, 27 Apr. 2022, [Peer-reviewed]
    Scientific journal
  • Low-Cost Cell-Surface-Mimic Analysis of Ligand Interactions of Biotinylated Immune Receptors Using Surface Plasmon Resonance.
    Kimiko Kuroki; Hideo Fukuhara; Takashi Tadokoro; Katsumi Maenaka
    Methods in molecular biology (Clifton, N.J.), 2421, 21, 35, 2022, [International Magazine]
    English, Scientific journal, On the immune cell surface, many immune receptors are expressed and modulate the inhibitory or activating signals to control the immune responses. Recently, some of these receptors have been categorized as immune checkpoint receptors and targeted for cancer immunity or autoimmune diseases. To analyze the weak and fast binding typical for immune receptor-ligand interactions, a real-time surface plasmon resonance (SPR) technique is useful. However, it sometimes becomes difficult to optimize the immobilization conditions and appropriate controls. Considering that receptor orientation is relevant for achieving function on the cell surface, it is important to immobilize ligand proteins using specific tags at the membrane proximal end to avoid steric hindrance and structural changes in specific binding regions. Here we introduce a sensor chip, Sensor Chip CAP (Cytiva), which enables reversible and orientation-controlled immobilization of biotinylated ligands, resulting in a significant cost-effective method. We further show preparation methods of several biotinylated immune receptor proteins for SPR analysis, which are also useful for structural and other functional analyses.
  • Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein.
    Kohei Yumoto; Tomoaki Arisaka; Kazuma Okada; Kyosuke Aoki; Toyoyuki Ose; Tatsunori Masatani; Makoto Sugiyama; Naoto Ito; Hideo Fukuhara; Katsumi Maenaka
    Viruses, 13, 11, 19 Nov. 2021, [International Magazine]
    English, Scientific journal, Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15-13 and 12-22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15-13 and 12-22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm-baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15-13 Fab interacts with the RABVG, with a KD value at the nM level, and that the 12-22 Fab has a weaker binding affinity (KD ~ μM) with the RABVG compared to the 15-13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15-13 and 12-22 antibodies as another potential biopharmaceutical for targeting rabies. The 15-13 and 12-22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the μM level of the KD, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.
  • A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site.
    Taishi Onodera; Shunsuke Kita; Yu Adachi; Saya Moriyama; Akihiko Sato; Takao Nomura; Shuhei Sakakibara; Takeshi Inoue; Takashi Tadokoro; Yuki Anraku; Kohei Yumoto; Cong Tian; Hideo Fukuhara; Michihito Sasaki; Yasuko Orba; Nozomi Shiwa; Naoko Iwata; Noriyo Nagata; Tateki Suzuki; Jiei Sasaki; Tsuyoshi Sekizuka; Keisuke Tonouchi; Lin Sun; Shuetsu Fukushi; Hiroyuki Satofuka; Yasuhiro Kazuki; Mitsuo Oshimura; Tomohiro Kurosaki; Makoto Kuroda; Yoshiharu Matsuura; Tadaki Suzuki; Hirofumi Sawa; Takao Hashiguchi; Katsumi Maenaka; Yoshimasa Takahashi
    Immunity, 54, 10, 2385, 2398, 12 Oct. 2021, [International Magazine]
    English, Scientific journal, Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.
  • The Physiological TMPRSS2 Inhibitor HAI-2 Alleviates SARS-CoV-2 Infection.
    Yuriko Tomita; Shutoku Matsuyama; Hideo Fukuhara; Katsumi Maenaka; Hiroaki Kataoka; Takao Hashiguchi; Makoto Takeda
    Journal of virology, 95, 12, 24 May 2021, [International Magazine]
    English
  • 【ウイルス感染症に取り組む生物物理学】ウイルス生物物理学 創薬モダリティへの貢献
    前仲 勝実; 福原 秀雄; 橋口 隆生; Caaveiro Jose M.M.; 長門石 曉; 黒田 大祐; 津本 浩平
    生物物理, 61, 2, 082, 089, (一社)日本生物物理学会, Mar. 2021
    Japanese
  • Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G isoforms
    黒木, 喜美子; 松原, 永季; 神田, 諒; 宮下, 尚之; 白石, 充典; 福永, 裕子; 上敷領, 淳; 福永, 淳; 福原, 秀雄; 廣瀬, 薫; Hunt, Joan S; 杉田, 有治; 喜多, 俊介; 尾瀬, 農之; 前仲, 勝実
    福山大学薬学部研究年報 = Annual report of the Faculty of Pharmacy & Pharmaceutical Sciences, Fukuyama University, 38, 35, 36, 福山大学薬学部, 25 Dec. 2020
    Japanese, Research institution
  • LILRA2のリガンド認識機構の解明
    古川 敦; 山崎 莉佳; Jiaqi Wang; 平安 恒幸; 湯本 航平; 福原 秀雄; 荒瀬 尚; 前仲 勝実
    日本生化学会大会プログラム・講演要旨集, 93回, [2Z13, 659)], (公社)日本生化学会, Sep. 2020
    Japanese
  • Molecular mechanism of the recognition of bacterially cleaved immunoglobulin by the immune regulatory receptor LILRA2.
    Rika Yamazaki; Atsushi Furukawa; Kouyuki Hirayasu; Kohei Yumoto; Hideo Fukuhara; Hisashi Arase; Katsumi Maenaka
    The Journal of biological chemistry, 295, 28, 9531, 9541, 10 Jul. 2020, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Human leukocyte immunoglobulin-like receptors (LILRs) typically regulate immune activation by binding to the human leukocyte antigen class I molecules. LILRA2, a member of the LILR family, was recently reported to bind to other unique ligands, the bacterially degraded Igs (N-truncated Igs), for the activation of immune cells. Therefore, LILRA2 is currently attracting significant attention as a novel innate immune receptor. However, the detailed recognition mechanisms required for this interaction remain unclear. In this study, using several biophysical techniques, we uncovered the molecular mechanism of N-truncated Ig recognition by LILRA2. Surface plasmon resonance analysis disclosed that LILRA2 specifically binds to N-truncated Ig with weak affinity (Kd = 4.8 μm) and fast kinetics. However, immobilized LILRA2 exhibited a significantly enhanced interaction with N-truncated Ig due to avidity effects. This suggests that cell surface-bound LILRA2 rapidly monitors and identifies bi- or multivalent abnormal N-truncated Igs through specific cross-linking to induce immune activation. Van't Hoff analysis revealed that this interaction is enthalpy-driven, with a small entropy loss, and results from differential scanning calorimetry indicated the instability of the putative LILRA2-binding site, the Fab region of the N-truncated Ig. Atomic force microscopy revealed that N truncation does not cause significant structural changes in Ig. Furthermore, mutagenesis analysis identified the hydrophobic region of LILRA2 domain 2 as the N-truncated Ig-binding site, representing a novel ligand-binding site for the LILR family. These results provide detailed insights into the molecular regulation of LILR-mediated immune responses targeting ligands that have been modified by bacteria.
  • Structural characteristics of measles virus entry.
    Hideo Fukuhara; Mwila Hilton Mwaba; Katsumi Maenaka
    Current opinion in virology, 41, 52, 58, Apr. 2020, [International Magazine]
    English, Scientific journal, Measles virus, a member of the genus Morbillivirus, is highly contagious and still shows considerable mortality with over 100000 deaths annually, although efficient attenuated vaccines exist. Recent studies of measles virus haemagglutinin (MeV-H) and its receptor, including crystallographic and electron microscopic structural analyses combined with functional assays, have revealed how the MeV-H protein recognizes its cognate receptors, SLAM and Nectin-4, and how the glycan shield ensures effective vaccination. In addition, the crystal structure of the MeV-F protein indicated its similarity to those of other paramyxoviruses. Taking into account these data, several models of viral entry/membrane fusion of measles viruses and related paramyxoviruses have been proposed. Furthermore, anti-MeV-F inhibitors targeted to specific regions to inhibit MeV-F protein activation were reported, with potency for preventing MeV infection. The inhibitors targeted for entry events may potentially be applied to treatment of MeV-derived diseases, although escape mutations and drug profiles should be considered.
  • Measles Virus Hemagglutinin Protein Establishes a Specific Interaction With the Extreme N-Terminal Region of Human Signaling Lymphocytic Activation Molecule to Enhance Infection.
    Fumio Seki; Yuta Yamamoto; Hideo Fukuhara; Kazue Ohishi; Tadashi Maruyama; Katsumi Maenaka; Hiroaki Tokiwa; Makoto Takeda
    Frontiers in microbiology, 11, 1830, 1830, 2020, [International Magazine]
    English, Scientific journal, Measles virus (MV) is a human pathogen that is classified in the genus Morbillivirus in the family Paramyxoviridae together with several non-human animal morbilliviruses. They cause severe systemic infections by using signaling lymphocytic activation molecule (SLAM) and poliovirus receptor-like 4 expressed on immune and epithelial cells, respectively, as receptors. The viral hemagglutinin (H) protein is responsible for the receptor-binding. Previously determined structures of MV-H and SLAM complexes revealed a major binding interface between the SLAM V domain and MV-H with four binding components (sites 1-4) in the interface. We studied the MV-H and human SLAM (hSLAM) complex structure in further detail by in silico analyses and determined missing regions or residues in the previously determined complex structures. These analyses showed that, in addition to sites 1-4, MV-H establishes a unique interaction with the extreme N-terminal region (ExNTR) of hSLAM. The first principles calculation-based fragment molecular orbital computation method revealed that methionine at position 29 (hSLAM-Met29) is the key residue for the interaction. hSLAM-Met29 was predicted to establish a CH-π interaction with phenylalanine at position 549 of MV-H (MVH-Phe549). A cell-cell fusion assay showed that the hSLAM-Met29 and MVH-Phe549 interaction is important for hSLAM-dependent MV membrane fusion. Furthermore, Jurkat cell lines expressing hSLAM with or without Met29 and recombinant MV possessing the H protein with or without Phe549 showed that the hSLAM-Met29 and MVH-Phe549 interaction enhanced hSLAM-dependent MV infection by ~10-fold. We speculate that in the evolutionary history of morbilliviruses, this interaction may have contributed to MV adaptation to humans because this interaction is unique for MV and only MV uses hSLAM efficiently among morbilliviruses.
  • Biophysical characterization and single-chain Fv construction of a neutralizing antibody to measles virus.
    Takashi Tadokoro; Mst Lubna Jahan; Yuri Ito; Maino Tahara; Surui Chen; Atsutoshi Imai; Natsumi Sugimura; Koki Yoshida; Mizuki Saito; Toyoyuki Ose; Takao Hashiguchi; Makoto Takeda; Hideo Fukuhara; Katsumi Maenaka
    The FEBS journal, 287, 1, 145, 159, Jan. 2020, [Peer-reviewed], [International Magazine]
    English, The measles virus (MV) is a major cause of childhood morbidity and mortality worldwide. We previously established a mouse monoclonal antibody, 2F4, which shows high neutralizing titers against eight different genotypes of MV. However, the molecular basis for the neutralizing activity of the 2F4 antibody remains incompletely understood. Here, we have evaluated the binding characteristics of a Fab fragment of the 2F4 antibody. Using the MV infectious assay, we demonstrated that 2F4 Fab inhibits viral entry via either of two cellular receptors, SLAM and Nectin4. Surface plasmon resonance (SPR) analysis of recombinant proteins indicated that 2F4 Fab interacts with MV hemagglutinin (MV-H) with a KD value at the nm level. Furthermore, we designed a single-chain Fv fragment of 2F4 antibody as another potential biopharmaceutical to target measles. The stable 2F4 scFv was successfully prepared by the refolding method and shown to interact with MV-H at the μm level. Like 2F4 Fab, scFv inhibited receptor binding and viral entry. This indicates that 2F4 mAb uses the receptor-binding site and/or a neighboring region as an epitope with high affinity. These results provide insight into the neutralizing activity and potential therapeutic use of antibody fragments for MV infection.
  • Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.
    Kimiko Kuroki; Haruki Matsubara; Ryo Kanda; Naoyuki Miyashita; Mitsunori Shiroishi; Yuko Fukunaga; Jun Kamishikiryo; Atsushi Fukunaga; Hideo Fukuhara; Kaoru Hirose; Joan S Hunt; Yuji Sugita; Shunsuke Kita; Toyoyuki Ose; Katsumi Maenaka
    Journal of immunology (Baltimore, Md. : 1950), 203, 12, 3386, 3394, 15 Dec. 2019, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the β2-microglobulin (β2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized β2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with β2m, thus accounting for β2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.
  • Specificity of Morbillivirus Hemagglutinins to Recognize SLAM of Different Species.
    Hideo Fukuhara; Yuri Ito; Miyuki Sako; Mizuho Kajikawa; Koki Yoshida; Fumio Seki; Mwila Hilton Mwaba; Takao Hashiguchi; Masa-Aki Higashibata; Toyoyuki Ose; Kimiko Kuroki; Makoto Takeda; Katsumi Maenaka
    Viruses, 11, 8, 19 Aug. 2019, [Peer-reviewed], [International Magazine]
    English, Measles virus (MV) and canine distemper virus (CDV) are highly contagious and deadly, forming part of the morbillivirus genus. The receptor recognition by morbillivirus hemagglutinin (H) is important for determining tissue tropism and host range. Recent reports largely urge caution as regards to the potential expansion of host specificities of morbilliviruses. Nonetheless, the receptor-binding potential in different species of morbillivirus H proteins is largely unknown. Herein, we show that the CDV-H protein binds to the dog signaling lymphocyte activation molecule (SLAM), but not to the human, tamarin, or mouse SLAM. In contrast, MV-H can bind to human, tamarin and dog SLAM, but not to that of mice. Notably, MV binding to dog SLAM showed a lower affinity and faster kinetics than that of human SLAM, and MV exhibits a similar entry activity in dog SLAM- and human SLAM-expressing Vero cells. The mutagenesis study using a fusion assay, based on the MV-H-SLAM complex structure, revealed differences in tolerance for the receptor specificity between MV-H and CDV-H. These results provide insights into H-SLAM specificity related to potential host expansion.
  • A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells
    Yoichiro Fujioka; Shinya Nishide; Toyoyuki Ose; Tadaki Suzuki; Izumi Kato; Hideo Fukuhara; Mari Fujioka; Kosui Horiuchi; Aya O. Satoh; Prabha Nepal; Sayaka Kashiwagi; Jing Wang; Mika Horiguchi; Yuko Sato; Sarad Paudel; Asuka Nanbo; Tadaaki Miyazaki; Hideki Hasegawa; Katsumi Maenaka; Yusuke Ohba
    Cell Host and Microbe, 23, 6, 809, 818.e5, Cell Press, 13 Jun. 2018, [Peer-reviewed]
    English, Scientific journal
  • The Role of Heparan Sulfate Proteoglycans as an Attachment Factor for Rabies Virus Entry and Infection.
    Sasaki M; Anindita PD; Ito N; Sugiyama M; Carr M; Fukuhara H; Ose T; Maenaka K; Takada A; Hall WW; Orba Y; Sawa H
    The Journal of infectious diseases, 217, 11, 1740, 1749, May 2018, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Rabies virus (RABV) is the causative agent of fatal neurological disease. Cellular attachment is the initial and essential step for viral infections. Although extensive studies have demonstrated that RABV uses various target cell molecules to mediate infection, no specific molecule has been identified as an attachment factor for RABV infection. Here we demonstrate that cellular heparan sulfate (HS) supports RABV adhesion and subsequent entry into target cells. Enzymatic removal of HS reduced cellular susceptibility to RABV infection, and heparin, a highly sulfated form of HS, blocked viral adhesion and infection. The direct binding between RABV glycoprotein and heparin was demonstrated, and this interaction was shown to require HS N- and 6-O-sulfation. We also revealed that basic amino acids in the ectodomain of RABV glycoprotein serve as major determinants for the RABV-HS interaction. Collectively, our study highlights a previously undescribed role of HS as an attachment factor for RABV infection.
  • Establishment of the BacMam system using silkworm baculovirus
    Atsutoshi Imai; Takashi Tadokoro; Shunsuke Kita; Masataka Horiuchi; Hideo Fukuhara; Katsumi Maenaka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 478, 2, 580, 585, Sep. 2016, [Peer-reviewed]
    English, Scientific journal
  • Structural aspects of C-type lectin receptors
    Atsushi Furukawa; Shunsuke Kita; Takashi Tadokoro; Hideo Fukuhara; Katsumi Maenaka
    C-Type Lectin Receptors in Immunity, 179, 190, Springer Japan, 01 Jan. 2016, [Peer-reviewed]
    English, In book
  • Crystal structure of extracellular domain of human lectin-like transcript 1 (LLT1), the ligand for natural killer receptor-P1A
    Shunsuke Kita; Haruki Matsubara; Yoshiyuki Kasai; Takaharu Tamaoki; Yuki Okabe; Hideo Fukuhara; Jun Kamishikiryo; Elena Krayukhina; Susumu Uchiyama; Toyoyuki Ose; Kimiko Kuroki; Katsumi Maenaka
    EUROPEAN JOURNAL OF IMMUNOLOGY, 45, 6, 1605, 1613, Jun. 2015, [Peer-reviewed]
    English, Scientific journal
  • New Binding Face of C-type Lectin-like Domains
    Hideo Fukuhara; Atsushi Furukawa; Katsumi Maenaka
    STRUCTURE, 22, 12, 1694, 1696, Dec. 2014, [Peer-reviewed]
    English
  • 1P005 Structure analysis of lectin-like transcript 1 (LLT1) and model building of LLT1-CD161 complex(Protein: Structure,Poster,The 52th Annual Meeting of the Biophysical Society of Japan(BSJ2014))
    Shunsuke Kita; Haruki Matsubara; Jun Kamishikiryo; Yuki Okabe; Hideo Fukuhara; Kimiko Kuroki; Katsumi Maenaka
    Seibutsu Butsuri, 54, 1, S141, The Biophysical Society of Japan General Incorporated Association, 2014
    English
  • TMPRSS2 Is an Activating Protease for Respiratory Parainfluenza Viruses
    Masako Abe; Maino Tahara; Kouji Sakai; Hiromi Yamaguchi; Kazuhiko Kanou; Kazuya Shirato; Miyuki Kawase; Masahiro Noda; Hirokazu Kimura; Shutoku Matsuyama; Hideo Fukuhara; Katsumi Mizuta; Katsumi Maenaka; Yasushi Ami; Mariko Esumi; Atsushi Kato; Makoto Takeda
    JOURNAL OF VIROLOGY, 87, 21, 11930, 11935, Nov. 2013, [Peer-reviewed]
    English, Scientific journal
  • Entry Mechanism of Morbillivirus Family
    Hideo Fukuhara; Surui Chen; Shin Takeda; Katsumi Maenaka
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN, 133, 5, 549, 559, May 2013, [Peer-reviewed]
    Japanese
■ Other Activities and Achievements
■ Syllabus
  • 一般教育演習(ﾌﾚｯｼｭﾏﾝｾﾐﾅｰ), 2024年, 学士課程, 全学教育
■ Research Themes
  • 非病原性フラビウイルスを用いた広域交差性エピトープの解析とワクチン開発
    科学研究費助成事業
    01 Apr. 2023 - 31 Mar. 2027
    大場 靖子; 福原 秀雄
    ISFV-IIに対するモノクローナル抗体作製のためのマウス免疫用の抗原として、ISFV-IIであるPsorophora virus（PSFV）を蚊培養細胞であるC6/36細胞を用いて増殖させ、20％ショ糖クッション法にてウイルスの濃縮を行い抗原とした。続いて、抗原を免疫したマウスの脾臓から、抗原特異的B細胞受容体を発現している胚中心由来B細胞をソーティングにより単離するためのプローブの作出を試みた。PSFVのCapsid（C）、precursor membrane（prM）並びにEnvelope（E）タンパク質から構成されるウイルス様粒子（VLP）の発現コンストラクトを作出し、哺乳類由来培養細胞を用いて発現させたVLPに蛍光標識することでプローブとした。また、胚中心由来B細胞をソーティングするための方法の最適化、また単離したB細胞の単細胞培養の方法を最適化した。フラビウイルスの中和試験並びにADE試験を迅速且つ高感度に判定するために、ウイルスを用いた試験法の代替試験法として、一回感染粒子（SRIP）を用いた試験の構築を試みた。SRIP作製のために、West Nile virus（WNV）由来のRepliconを発現するプラスミド、WNV由来のCタンパク質、並びに各フラビウイルスのprMEを発現するプラスミドを作出した。これらのプラスミドを哺乳類培養細胞に共発現させることでSRIPを作出し、中和試験法、並びにADE試験法を構築した。また、クライオ電子顕微鏡を用いてISFV-IIの構造解析を実施するためのウイルスを精製した。ショ糖クッション法にて濃縮したPSFVを密度勾配遠心法にて密度別にフラクションを回収し、PSFV抗原陽性だったフラクションを限外濾過膜にて濃縮した。PSFV抗原用性フラクションをさらにショ糖密度勾配遠心法によって精製したサンプルについて現在解析を進めている。
    日本学術振興会, 基盤研究(B), 北海道大学, 23K27831
  • Inhibitor development of Pseudomonas aeruginosa MurD using a novel molecular design method targeting the conformation changing process.
    Grants-in-Aid for Scientific Research
    01 Apr. 2018 - 31 Mar. 2021
    Fukuhara Hideo
    MurD, which is one of the cell wall synthases, is a promising target for antibacterial drug agents against Pseudomonas aeruginosa. While MurD recognizes substrates specifically, no inhibitors with sufficient efficacy have been developed. In this study, we have determined the crystal structure of Pseudomonas aeruginosa MurD. Based on this, some inhibitors were designed and evaluated using MD simulation, enzymatic assay, and binding analysis. As a result, we found a promising lead compound showing lower IC50 than known inhibitors.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 18K06569
  • Analysis of respiratory virus activating protease TMPRSS2 and its inhibitor compounds
    Grants-in-Aid for Scientific Research
    01 Apr. 2018 - 31 Mar. 2021
    Takeda Makoto
    We have previously shown that the host protease TMPRSS2 activates a wide variety of respiratory viruses and is responsible for the activation of influenza viruses in vivo. In the present study, we analyzed the activation of SARS-CoV-2 by TMPRSS2 and the inhibition of TMPRSS2 and SARS-CoV-2 infection by HAI-2. SARS-CoV-2 infection was markedly enhanced by TMPRSS2 expression. On the other hand, TMPRSS2-mediated SARS-CoV-2 infection was inhibited by HAI-2 in a dose-dependent manner. Knockdown of HAI-2 from cells resulted in a 10-fold increase in the number of viral genome copies detected
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), National Institute of Infectious Diseases, 18H02665
  • Molecular basis for cell entry of canine distemper virus
    Grants-in-Aid for Scientific Research
    01 Apr. 2015 - 31 Mar. 2018
    MAENAKA Katsumi
    The structural features of the membrane fusion protein (CDV-F) of canine distemper virus (CDV) were observed by cryo-electron microscopy. The sample preparation and grid manufacturing conditions were successfully optimized for high-resolution analysis. On the other hand, for the receptor-binding protein (CDV-H) of the same virus, several anti-CDV-H monoclonal antibodies were setablished by rat intestinal lymph node method. The subtypes and CDV-H binding properties of the obtained antibodies were examined. The membrane fusion assay showed that each antibody has different binding affinity and specificity. These results give insight on the molecular basis of viral entry and vaccine effectiveness of CDV and related viruses.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 15H02384