中和単クローン抗体パネルを用いたＨＩＶ感染の治癒に向けた研究

2014 Fiscal Year Annual Research Report

• Project/Area Number: 24591484
• Research Institution: Kumamoto University
• Principal Investigator: 松下 修三 熊本大学, エイズ学研究センター, 教授 (00199788)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Keywords: 中和抗体 / ウイルス / 動物モデル

Outline of Annual Research Achievements

抗体の可変部位を分子クローニングする新しい方法を用いて、HIV-1非サブタイプB感染症例から、新規単クローン抗体を分離し、有効なAIDSワクチン開発に貢献すること、および、cART治療下に残存するHIV-1産生細胞を排除する治療戦略の基礎研究として、抗体依存性細胞傷害活性（ADCC）に関して臨床分離株を標的としたアッセイシステムを構築し、有効な抗体反応を同定することが本研究の目的である。
1．昨年までの研究で、広範囲の中和抗体活性を示すCRF01_AE感染症例を同定した。該当症例の末梢血単核球からメモリーB細胞を分離、EBVで形質転換後にクローニングした。培養上清をCRF01_AE envelope発現細胞にてスクリーニングし、特異的結合活性が見られたクローンについてsubtype AE, A, B及びCへの交差反応性を検討し、候補となった複数のクローンからRNAを抽出し、Oligo(dT)プライマーと逆転写酵素でcDNAを作製した。これを鋳型として抗体の重鎖、軽鎖のV領域を特異的に増幅後クローニングし、組み換え抗体を作成して、活性の測定とエピトープの同定を行っている。
2． ADCC活性に関しては、サブタイプB感染例由来の抗体パネルを用いて検討した。SF2,BaL,YU2など研究室で用いられる株ばかりでなく、5種類の初感染株（transmitted /founder virus）を用いspinoculationという方法で、CEM-NKR 細胞にHIVを感染させ標的細胞とした。これらの臨床分離株に対するADCC活性を調べると、我々が作成した12種類の抗体パネルの中のどれかが必ずADCC活性を示し、ポリクローナルな抗体反応が重要であることを明らかにした。

Research Products

• [Journal Article] Passive transfer of neutralizing monoclonal antibody KD-247reduces plasma viral load in patients chronically infected with HIV-1. (2015)
  • Author(s): Matsushita, S., Yoshimura, K., Ramirez, K.P., Pisupati, J., Murakami, T.
  • Journal Title: AIDS Volume: 29 Pages: 453-462
  • DOI: 10.1097/QAD.0000000000000570
  • Peer Reviewed
• [Journal Article] Complementary and synergistic activities of anti-V3, CD4bs and CD4i antibodies derived from a single individual can cover a wide range of HIV-1 strains. (2015)
  • Author(s): Ramirez, K.P., Kuwata, T., Maruta, Y, Tanaka K., Muntasir, A., Yoshimura, K., Matsushita, S.
  • Journal Title: Virology Volume: 475 Pages: 187-203
  • DOI: 10.1016/j.virol.2014.11.011
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247. (2015)
  • Author(s): Kirby, K.A., Ong Y.T., Hachiya, A., Laughlin, T.G.,Chiang, L.A., Pan Y., Moran, J.L., Marchand, B.,Singh, K., Gallazzi, F., Quinn, T.P., Yoshimura, K., Murakami, T., Matsushita S., Sarafianos, S.G.
  • Journal Title: The FASEB Journal Volume: 29 Pages: 1-11
  • DOI: 10.1096/fj.14-252262
  • Peer Reviewed / Open Access
• [Journal Article] Impact of maraviroc-resistant and low-CCR5- adapted mutations induced by in vitro passage on sensitivity to anti-envelope neutralizing antibodies. (2014)
  • Author(s): Yoshimura, K., Harada, S., Boonchawalit, S., Kawanami, Y., Matsushita, S.
  • Journal Title: J. Gen. Virol. Volume: 95 Pages: 1816-1826
  • DOI: 10.1099/vir.0.062885-0
  • Peer Reviewed
• [Presentation] Enhanced Neutralization of HIV-1 with Fusion Inhibitor Resistant Mutations. The annual Conference on Retroviruses and Opportunistic Infections (CROI 2015). (2015)
  • Author(s): Muntasir, A., Kuwata, T., Ramirez, K., Maruta, Y., Tanaka K., Shimura, K., Oishi, S., Fujii, N., Matsuoka, M., Matsushita, S.
  • Organizer: The annual Conference on Retroviruses and Opportunistic Infections (CROI 2015).
  • Place of Presentation: the Washington State Convention Center(Seattle, Washington, USA.)
  • Year and Date: 2015-02-23 – 2015-02-26
• [Presentation] Therapeutic application of neutralizing antibodies based on the recent data on complementary and synergistic activities against a wide range of HIV-1 strains. (2015)
  • Author(s): Matsushita, S., Ramirez, K.P., Maruta, Y., Tanaka, K., Muntasir, A., Kuwata, T., Pisupait, J., Murakami, T., Yoshimura, K.
  • Organizer: the 17th International Conference on Emerging Infectious Diseases (EID) Emerging Infectious Diseases Meeting
  • Place of Presentation: Building of Humanities and Social Sciences, Academia Sinica(Taipei, Taiwan)
  • Year and Date: 2015-01-26 – 2015-01-27
• [Presentation] Mechanism of post-attachment neutralization of single-chain variable fragment (scFv) from anti-V3 monoclonal antibody with cross-reactivity. (2015)
  • Author(s): Maruta, Y., Kuwata, T., Tanaka, K., Nakahara,Y., Ramirez, K., Alam, M., Egami, Y., Hirata, I., Suwa, Y., Morioka, H., Matsushita, S.
  • Organizer: Korea-Japan Joint Seminar on HIV/AIDS.
  • Place of Presentation: 熊本大学山崎記念会館（熊本市）
  • Year and Date: 2015-01-10 – 2015-01-10
• [Presentation] Complementary effect of anti-V3, CD4bs and CD4i antibodies accounts for cross-neutralizing and non-neutralizing activities against HIV-1. (2014)
  • Author(s): Ramirez, K.P., Kuwata, T., Maruta, Y, Tanaka K., Muntasir, A., Yoshimura, K., Matsushita, S.
  • Organizer: 第28回日本エイズ学会学術集会・総会.
  • Place of Presentation: 大阪国際会議場（大阪市）
  • Year and Date: 2014-12-03 – 2014-12-05
• [Presentation] V3エピトープへの交叉反応性をもつ中和抗体の遺伝子組み換えによる小型化の試み (2014)
  • Author(s): 江上由華、丸田泰広、田中和樹、Muntasir A., Ramirez K., 桑田岳夫、松下修三.
  • Organizer: 第28回日本エイズ学会学術集会・総会.
  • Place of Presentation: 大阪国際会議場（大阪市）
  • Year and Date: 2014-12-03 – 2014-12-05
• [Presentation] Mechanism of post-attachment neutralization of single-chain variable fragment (scFv) from anti-V3 monoclonal antibody against HIV-1 (2014)
  • Author(s): Maruta, Y., Kuwata, T., Tanaka, K., Nakahara,Y., Ramirez, K., Alam, M., Egami, Y., Hirata I., Suwa, Y., Morioka, H., Matsushita, S.
  • Organizer: HIV R4P 2014
  • Place of Presentation: Cape Town International Convention Centre(Cape Town, South Africa)
  • Year and Date: 2014-10-28 – 2014-10-31
• [Presentation] Therapeutic application of neutralizing antibodies based on the recent data on complementary and synergistic activities against a wide range of HIV-1 strains. (2014)
  • Author(s): Matsushita, S., Ramirez, K.P., Maruta, Y., Tanaka, K., Muntasir, A., Kuwata, T., Pisupait, J., Murakami, T., Yoshimura, K.
  • Organizer: 15th Kumamoto AIDS Seminar.
  • Place of Presentation: ホテル日航熊本（熊本市）
  • Year and Date: 2014-10-01 – 2014-10-03
• [Presentation] Evaluation of complementarity and synergy of conventional anti-HIV-1 antibodies derived from a single individual (2014)
  • Author(s): Ramirez, K.P., Kuwata, T., Maruta, Y, Tanaka, K., Muntasir, A., Yoshimura, K., Matsushita, S.
  • Organizer: 15th Kumamoto AIDS Seminar
  • Place of Presentation: グランヴィリオホテル（阿蘇市）
  • Year and Date: 2014-10-01 – 2014-10-03
• [Presentation] Mutations in gp41 that confer resistance to fusion inhibitors enhance the neutralization sensitivity to antibodies against both gp41 and gp120. (2014)
  • Author(s): Muntasir, A., Kuwata, T., Ramirez, K., Enomoto, I., Maruta, Y., Tanaka, K., Rahman, K., Nakahara, Y., Egami, Y., Kawanami, Y., Murayama, H., Shimura, K., Matsuoka, M., Matsushita, S.
  • Organizer: 15th Kumamoto AIDS Seminar
  • Place of Presentation: グランヴィリオホテル（阿蘇市）
  • Year and Date: 2014-10-01 – 2014-10-03
• [Presentation] Mechanism of post-attachment neutralization of single-chain variable fragment (scFv) from anti-V3 monoclonal antibody against HIV-1. (2014)
  • Author(s): Maruta, Y., Kuwata, T., Tanaka, K., Nakahara,Y., Ramirez, K., Alam, M., Egami, Y., Hirata I., Suwa, Y., Morioka, H., Matsushita, S.
  • Organizer: 15th Kumamoto AIDS Seminar.
  • Place of Presentation: グランヴィリオホテル（阿蘇市）
  • Year and Date: 2014-10-01 – 2014-10-03
• [Presentation] Construction of neutralizing antibody fragment against V3 with a broad cross-reactivity. (2014)
  • Author(s): Tanaka K., Kuwata, T., Maruta, Y., Ramirez, K., Muntasir, A., Egami, Y., Enomoto, I., Kawanami, Y., Matsushita, S.
  • Organizer: 15th Kumamoto AIDS Seminar
  • Place of Presentation: グランヴィリオホテル（阿蘇市）
  • Year and Date: 2014-10-01 – 2014-10-03
• [Presentation] Passive transfer of neutralizing monoclonal antibody KD-247 reduces plasma viral load in patientschronically infected with HIV-1: a phase-1b clinical study of a humanized monoclonal antibody KD-247 (KD-1002). (2014)
  • Author(s): Matsushita, S., Yoshimura, K., Maeda, T, Murakami, T. KD-1002 Principal Investigators and The Protocol Team of Quintiles.
  • Organizer: 7th IAS Conference on HIV Pathogenesis, Treatment and Prevention
  • Place of Presentation: Kuala Lumpur Convention Centre (Kuala Lumpur, Malaysia)
  • Year and Date: 2014-06-30 – 2014-07-03
• [Presentation] Passive transfer of neutralizing monoclonal antibody KD-247 reduces plasma viral load in patients chronically infected with HIV-1：A Phase-1b clinical study of a humanized monoclonal antibody KD-247 (KD-1002) (2014)
  • Author(s): Matsushita, S.
  • Organizer: IAS Towards an HIV Cure Symposium
  • Place of Presentation: The Banquet Hall, Rits Carlton Hotel(Kuala Lumpur, Malaysia)
  • Year and Date: 2014-06-29 – 2014-06-30
• [Remarks] 熊本大学エイズ学研究センター松下プロジェクト研究室
  • URL: http://matsushita-lab.kumamoto-univ.jp