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cDNA cloning of Fab fragment against hepatitis C virus to produce humanized monoclonal antibody

Research Project

Project/Area Number 05454185
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Experimental pathology
Research InstitutionNihon University

Principal Investigator

ESUMI Mariko  Nihon University, School of Medicine, Associate, Professor, 医学部, 助教授 (30167291)

Co-Investigator(Kenkyū-buntansha) SUGITANI Masohiko  Nihon University, School of Medicine, Assistant Professor, 医学部, 講師 (40187654)
中林 啓記  日本大学, 医学部, 助手 (50237369)
Project Period (FY) 1993 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1995: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1994: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1993: ¥2,800,000 (Direct Cost: ¥2,800,000)
KeywordsHepatitis C virus / Monoclonal antibody / cDNA cloning / Neutralizing antibody / C型肝炎ウィルス
Research Abstract

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma in Japan. About 60% of HCV infection becomes persistent, resulting in development of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Neutralizing antibody against HCV is not clearly identified, because experimental systems of infection and proliferation of HCV are not yet established. Immune response analyzes in patients with acute and chronic hepatitis C suggest that neutralizing antibody is hardly induced in natural infection or a mutant of HCV escaping from neutralizing antibody generates by high mutation rate of HCV.In this work, neutralizing antibody is found out from animals such as mice and chimpanzee actively immunized with recombinant antigens, and is humanized by cDNA cloning of complimentarity-determining (CDR) of the neutralizing antibody.
We produced antibody against recombinant HCV envelope glycoproteins, E1 and E2, and synthesized hypervariable region (HVR) 1 peptides, as candidates for neutralizing antibody. Higher antibody response was induced in immunized mice and a chimpanzee, and immunoreactive peptides unique to animals were found in comparison with those of patients. Murine anti-E2 and anti-HVR1 antibodies but not anti-E1 antibody captured HCV.Chimpanzee antiserum against whole immunogens also captured HCV in vitro, and neutralized HCV infectivity in a chimpanzee. The immunized chimpanzee was also protected from HCV infection. Epitope analysis of capturing antibody showed that a reactive peptide exists in HVR1. Althogh HVR1 of HCV is isolate-specific, another HCV isolate was captured with these immunized sera. Now in order to clone cDNA of CDR of antibody, combinatorial antibody library is constructed from spleens of immunized mice and peripheral blood leukocytes of immunized chimpanzee.

Report

(4 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • 1993 Annual Research Report
  • Research Products

    (37 results)

All Other

All Publications (37 results)

  • [Publications] 林 仲信,他: "Molecular cloning and heterogeneity of the human hepatitis C virus (HCV) genome." J.Hepatol.17. s94-s107 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 野崎 周英,他: "Expression and epitope analysis of hepatitis C virus NS1" Int.Hepatol. Commun.1. 31-36 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 志方 俊夫,他: "C型肝炎ワクチン開発の展望." 治療. 75. 1215-1221 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 西原 司,他: "Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells." Gene. 129. 207-214 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 江角 真理子,他: "Hepatitis C virus and liver diseases." Pathol.Int.44. 85-95 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 平賀 正治,他: "Expression of HCV core protein in Escherichia coli and the establishment of ELISA system." Int.Hepatol. Commun.2. 37-42 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 江角 真理子: "C型肝炎." 薬局. 45. 657-662 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 江角 真理子,他: "C型肝炎ウイルスワクチン-開発は可能か-" nanoGIGA. 3. 995-1000 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 江角 真理子: "C型肝炎ウイスルとそのゲノタイプ." 臨床成人病. 24. 701-708 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 大原 守貴,他: "Direct PCR of whole blood and hair shafts by microwave treatment." Biotechniques. 17. 726-728 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 江角 真理子: "遺伝子操作と疾患モデル「トランスジェニック動物」." 産科と婦人科. 61. 1659-1665 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 加藤 珠実,他: "トランスジェニックマウスにおけるC型肝炎ウイルス(HCV)導入遺伝子の発現抑制とそのメチル化について." 日本臨床. 53. s92-s99 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Mamun Ahmed,他: "Murine hemoral immune response against recombinant structural proteins of hepatitis C virus distinct from those of patients." Microbiol. Immunol.42. 169-176 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 加藤 珠実,他: "Inactivation of hepatitis C virus cDNA transgene by hypermethylation in transgenic mice." Arch. Virol.114(印刷予定). (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 江角 真理子,他: "Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and by DNA amplitication-restricted transcription-translation system." J.Virol. Meth.(印刷予定). (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Hayashi N., Higashi H., Kaminaka K., Sugimoto H., Esumi M., Komatsu K., Hayashi K., Sugitani M., Suzuki K., Okano T., Nozaki C., MizunoK.and ShikataT.: "Molecular cloning and heterogeneity of the human hepatitis C virus (HCV) genome." J.Hepatol.17. s94-s107 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Nozaki C., Nishihara T., Makizumi K., Hoshiko K., Hamada F., Hayashi N., Esumi M., Shikata T.and Mizuno K.: "Expression and epitope analysis of hepatitis C virus NSI" Int.Hepatol.Commun.1. 31-36 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Nishihara T., Nozaki C., Nakatake H., Hoshiko K., Esumi M., Hayashi N., Hino K., Hamada F., Mizuno K., and Shikata T.: "Secretion and purification of hepatitis C virus NSI glycoprotein pruduced by recombinant baculovirus-infected insect cells." Gene. 129. 207-214 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Esumi M., and Shikata T.: "Hepatitis C virus and liver diseases." Pathol.Int.44. 85-95 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Hiraga M., Hayashi N., Nishizono A., Komatsu K., Moriyama M., Esumi M., Suzuki K., Kawahara T., Mizuno K., and Shikata T.: "Expression of HCV core protein in Escherichia coli and the establishment of ELISA system." Int.Hepatol.Commun.2. 37-42 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ohhara M., Kurosu Y., and Esumi M.: "Direct PCR of whole blood and hair shafts by microwave treatment" Biotechniques. 17 (4). 726-728 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ahmed M., Shikata T., and Esumi M.: "Murine humoral immune response against recompinant structural proteins of hepatitis C virus distinct from those of patients." Microbiol.Immunol.42(2). 169-176 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kato T., Ahmed M., Yamamoto T., Takahashi H., Oohara M., Ikeda T., Aida Y., Katsuki M., Arakawa Y., Shikata T., and Esumi M.: "Inactivation of hepatitis C virus cDNA transgene by hypermethylation in transgenic mice." Arch.Virol.114 (in press). (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Esumi M., Hayashi N., Takahashi H., Shikata T., Eto T., Nishihara T., Nozaki C., and Mizuno K.: "Immunoreactive core peptides of hepatitis C virus produced in Esherichia coli and by DNA amplification-restricted transcription-translation system." J.Virol.Meth. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 加藤珠実 他: "トランスジェニックマウスにおけるC型肝炎ウイルス(HCV)導入遺伝子の発現抑制とそのメチル化について." 日本臨床. 53. 92-99 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Mamun Ahmed 他: "Murine humoral immune response against recombinant structural proteins of hepatitis C virus distinct from those of patients." Microbiol. Immunol.42. 169-176 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] 加藤珠実 他: "Inactivation of hepatitis C virus cDNA transgene by hypermethylation in transgenic mice." Arch. Virol.141. (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] 江角真理子 他: "Hepatitis C virus and liver diseases." Pathology International. 44. 85-95 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 平賀正治 他: "Expression of HCV core protein in Escherichia coli and the establishment of ELISA system." International Hepatology Communications. 2. 37-42 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 大原守貴 他: "Direct PCR of whole blood and hair shafts by microwave treatment." Biotechniques. 17. 726-728 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 江角真理子: "病気とくすり C型肝炎" 薬局. 45. 657-662 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 江角真理子 他: "C型肝炎ウイルスワクチン-開発は可能か-" nanoGIGA. 3. 995-1000 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 江角真理子: "C型肝炎ウイルスとそのゲノタイプ." 臨床成人病. 24. 701-708 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 野崎周英他: "Expression and epitope analysis of hepatitis C virus NS1." Int.Hepatol.Commun.1. 31-36 (1)

    • Related Report
      1993 Annual Research Report
  • [Publications] 西原司他: "Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells." Gene. 129. 207-214 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 志方俊夫他: "C型肝炎ワクチン開発の展望" 治療. 75. 1215-1221 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 江角真理子他: "Hepatitis C virus and liver diseases." Pathol.Int.44. 85-95 (1994)

    • Related Report
      1993 Annual Research Report

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Published: 1993-04-01   Modified: 2016-04-21  

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