We would like to briefly introduce our papers that have been recently published.

Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.
Sasaki T, Setthapramote C, Kurosu T, Nishimura M, Asai A, Omokoko MD, Pipattanaboon C, Pitaksajjakul P, Limkittikul K, Subchareon A, Chaichana P, Okabayashi T, Hirai I, Leaungwutiwong P, Misaki R, Fujiyama K, Ono K, Okuno Y, Ramasoota P, Ikuta K.
Antiviral Res. 2013 Jun;98(3):423-31.

  デング熱・デング出血熱は、フラビウイルス科のデングウイルスにより引き起こされる感染症である。蚊をベクターとして流行が引き起こされ、世界中で25億人に感染リスクがあるとされている。しかしながら、治療薬やワクチン等の予防法は存在せず、対症療法及び蚊の駆除を中心に対策が取られている。そこで、私たちは、タイ・マヒドン大学熱帯医学部との共同研究として、デングウイルス感染患者中に誘導される中和抗体に着目した抗体医薬の開発を目的として、抗デングヒト型単クローン抗体の作製を行った。デングウイルス感染タイ人患者由来の末梢血単核球及びフュージョンパートナー細胞であるSPYMEG細胞(MBL社)との細胞融合法により、136クローンの抗デングウイルスヒト型単クローン抗体産生ハイブリドーマの作出を行い、報告を行った(BBRC, 2012)。
  本論文においては、デングウイルスの4種類の血清型に対し、中和能を強く示していた17抗体を用いて、より詳細な解析の結果を報告した。17種類の内13種類の抗体はデングウイルスエンベロープ蛋白質の1st domain II領域を認識していた。また、全ての抗体が抗体依存性感染増強(ADE)を示したが、ADEの要因と考えられる抗体のFc領域を除去したF(ab’)2抗体は中和能を維持させたままADEを抑えることが確認された。サックリングマウスを用いたin vivo試験では、抗体投与群での生存率の上昇が認められた。



  The development of vaccination methods that can overcome the emergence of new-type influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure (Figs. A and B). Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets (Fig. C). Through site-directed mutagenesis, modified EGFP with an epitope-mimicking structure embedded on their surface was prepared. We could conveniently confirm the mutation site of this EGFP variant retained β-sheet structure, because any known fluorescent protein never emit fluorescence when its overall structure is incomplete (Fig. D). The EGFP variant differing from wild-type (parental) EGFP by only nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus (Fig. E left). Moreover, three of five mice immunized with the EGFP variant followed by booster with an equivalent mCherry variant acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage (Fig.E right). In contrast to conventional methods such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.Fig(inoue2013jbc)



The changing nature of avian influenza A virus (H5N1)
Yohei Watanabe, Madiha S. Ibrahim, Yasuo Suzuki and Kazuyoshi Ikuta
Trends in Microbiology, January 2012, Vol. 20, No.1

  Highly pathogenic avian influenza A virus subtype H5N1 has been endemic in some bird species since its emergence in 1996 and its ecology, genetics and antigenic properties have continued to evolve. This has allowed diverse virus strains to emerge in endemic areas with altered receptor specificity, including a new H5 sublineage with enhanced binding affinity to the human-type receptor. The pandemic potential of H5N1 viruses is alarming and may be increasing. We review here the complex dynamics and changing nature of the H5N1 virus that may contribute to the emergence of pandemic strains.
  Changing ecology and interspecies transmission of the H5N1 virus
Since 2007, virus persistence in a wide range of birds has allowed phenotypic diversification in endemic areas and has led to the emergence of a new H5 sublineage, expanding possibilities for viral transmission from birds to humans.



Development of two types of rapid diagnostic test kits to detect the hemagglutinin or nucleoprotein of the swine-origin pandemic influenza A virus H1N1.
Mizuike, R., Sasaki, T., Baba, K., Iwamoto, H., Shibai, Y., Kosaka, M., Kubota-Koketsu, R., Tang, C.S., Du, A., Sakudo, A., Tsujikawa, M., Yunoki, M., and Ikuta, K.
Clin. Vaccine Immunol., 2011 Mar;18(3):494-9.

  Since its emergence in April 2009, pandemic flu H1N1(H1N1 pdm), a new type of influenza A virus with a triple reassortant genome, has spreadt hroughout the world. Initial attempts to diagnose patients using immunochromatography(IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm, but not seasonal influenza A(H1NlandH3N2) or B viruses, Using two of these antibodies, one recognizing viral hemagglutinin(HA), the other nucleoprotein(NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illness. The specificity of both lC testkits was very high (93.0% for HA kit and lOO% for NP kit). The test sensitivities for detection of H1Nl pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A detection assay. Use of th eanti-NP antibody could allow for the rapid and accurate diagnosis of H1N1 pdm infections.



Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt.
Watanabe,Y., Ibrahim, M.S., Ellakany, H.F., Kawashita, N., Mizuike, R., Hiramatsu, H., Sriwilaijamen, N., Takagi, T., Suzuki, Y. and lkuta, K.
PLoS Pathog. 7, e1002068, 2011.

  Highly pathogenic avian influenza A virus, subtype H5N1 is currently wide spread in Asia, Europe, and Afirica. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide. In this study, we found that new H5 sublineages have acquired an enhanced binding affinity for human-type receptor during viral diversification in local, bird populations. This is the first report identifying amino acid changes in H5HA responsible for an increase in human H5Nl infections in an endemic area. Our findings may explain why Egypt has recently had the highest number of human H5 cases worldwide. This paper was published in PLoS Pathogens, and was also introduced as Featured Research on the Web(May 2011 Issue).




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