Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro

Douglas F. Lake, Takashi Kawamura, Takami Tomiyama, W. Edward Robinson, Yoh Ichi Matsumoto, Yasuhiko Masuho, Evan M Hersh

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Abstract

Objective: The purpose of this study was to develop and characterize human monoclonal antibodies (HuMAb) that neutralize HIV-1. Design: Based upon previous studies involving the generation of HuMAb that neutralize other enveloped viruses, we thought it feasible to generate HuMAb that might neutralize HIV-1. Methods: A HuMAb was generated by fusing splenic B-cells from an HIV-positive patient with a mouse myeloma cell line. Flow cytometry was used to determine surface reactivity of the HuMAb on HIV-infected and non-infected cells. Radioimmunoprecipitation was employed to elucidate the antigen recognized by the HuMAb. A cell survival assay was used to determine the ability of the HuMAb to neutralize divergent isolates of HIV-1 in the presence or absence of complement. A gp120-CD4 inhibition enzyme-linked immunosorbent assay (ELISA) was developed in order to initiate studies to determine the mechanism of neutralization by the HuMAb. Results: An anti-HIV HuMAb was generated that neutralized two HIV-1 isolates (IIIB and MN) without complement and which neutralized one divergent isolate (RF) and one clinical isolate in the presence of complement. This HuMAb, designated S1-1, was found, by flow cytometric analysis, to react with the surface of HIV-1-infected but not with uninfected cells. Radioimmunoprecipitation analysis demonstrated that S1-1 binds to native HIV gp120, but not dithiothreitol (DTT)-treated gp120. In addition, HuMAb S1-1 did not bind to denatured HIV antigens in Western blot analysis. HuMAb S1-1 effectively inhibited the binding of gp120 to soluble CD4 in ELISA. Conclusions: These results suggest that the epitope recognized by S1-1 is conformational and conserved among diverse HIV-1 isolates and may represent an uncharacterized HIV neutralizing domain within or close to the CD4 binding domain on gp120. HuMAb S1-1 might have a role to play in vaccine development or passive immunotherapy. Keywords: Anti-HIV human monoclonal antibodies, HIV neutralization, complement-dependent neutralization of HIV.

Original languageEnglish (US)
Pages (from-to)17-24
Number of pages8
JournalAIDS
Volume6
Issue number1
StatePublished - Jan 1992

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HIV-1
Monoclonal Antibodies
HIV
In Vitro Techniques
Radioimmunoprecipitation Assay
Enzyme-Linked Immunosorbent Assay
HIV Envelope Protein gp120
HIV Antigens
Passive Immunization
Dithiothreitol
Epitopes
Cell Survival
Flow Cytometry
B-Lymphocytes
Vaccines
Western Blotting
Viruses
Antigens
Cell Line

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Lake, D. F., Kawamura, T., Tomiyama, T., Robinson, W. E., Matsumoto, Y. I., Masuho, Y., & Hersh, E. M. (1992). Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro. AIDS, 6(1), 17-24.

Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro. / Lake, Douglas F.; Kawamura, Takashi; Tomiyama, Takami; Robinson, W. Edward; Matsumoto, Yoh Ichi; Masuho, Yasuhiko; Hersh, Evan M.

In: AIDS, Vol. 6, No. 1, 01.1992, p. 17-24.

Research output: Contribution to journalArticle

Lake, DF, Kawamura, T, Tomiyama, T, Robinson, WE, Matsumoto, YI, Masuho, Y & Hersh, EM 1992, 'Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro', AIDS, vol. 6, no. 1, pp. 17-24.
Lake DF, Kawamura T, Tomiyama T, Robinson WE, Matsumoto YI, Masuho Y et al. Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro. AIDS. 1992 Jan;6(1):17-24.
Lake, Douglas F. ; Kawamura, Takashi ; Tomiyama, Takami ; Robinson, W. Edward ; Matsumoto, Yoh Ichi ; Masuho, Yasuhiko ; Hersh, Evan M. / Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro. In: AIDS. 1992 ; Vol. 6, No. 1. pp. 17-24.
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abstract = "Objective: The purpose of this study was to develop and characterize human monoclonal antibodies (HuMAb) that neutralize HIV-1. Design: Based upon previous studies involving the generation of HuMAb that neutralize other enveloped viruses, we thought it feasible to generate HuMAb that might neutralize HIV-1. Methods: A HuMAb was generated by fusing splenic B-cells from an HIV-positive patient with a mouse myeloma cell line. Flow cytometry was used to determine surface reactivity of the HuMAb on HIV-infected and non-infected cells. Radioimmunoprecipitation was employed to elucidate the antigen recognized by the HuMAb. A cell survival assay was used to determine the ability of the HuMAb to neutralize divergent isolates of HIV-1 in the presence or absence of complement. A gp120-CD4 inhibition enzyme-linked immunosorbent assay (ELISA) was developed in order to initiate studies to determine the mechanism of neutralization by the HuMAb. Results: An anti-HIV HuMAb was generated that neutralized two HIV-1 isolates (IIIB and MN) without complement and which neutralized one divergent isolate (RF) and one clinical isolate in the presence of complement. This HuMAb, designated S1-1, was found, by flow cytometric analysis, to react with the surface of HIV-1-infected but not with uninfected cells. Radioimmunoprecipitation analysis demonstrated that S1-1 binds to native HIV gp120, but not dithiothreitol (DTT)-treated gp120. In addition, HuMAb S1-1 did not bind to denatured HIV antigens in Western blot analysis. HuMAb S1-1 effectively inhibited the binding of gp120 to soluble CD4 in ELISA. Conclusions: These results suggest that the epitope recognized by S1-1 is conformational and conserved among diverse HIV-1 isolates and may represent an uncharacterized HIV neutralizing domain within or close to the CD4 binding domain on gp120. HuMAb S1-1 might have a role to play in vaccine development or passive immunotherapy. Keywords: Anti-HIV human monoclonal antibodies, HIV neutralization, complement-dependent neutralization of HIV.",
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AU - Lake, Douglas F.

AU - Kawamura, Takashi

AU - Tomiyama, Takami

AU - Robinson, W. Edward

AU - Matsumoto, Yoh Ichi

AU - Masuho, Yasuhiko

AU - Hersh, Evan M

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N2 - Objective: The purpose of this study was to develop and characterize human monoclonal antibodies (HuMAb) that neutralize HIV-1. Design: Based upon previous studies involving the generation of HuMAb that neutralize other enveloped viruses, we thought it feasible to generate HuMAb that might neutralize HIV-1. Methods: A HuMAb was generated by fusing splenic B-cells from an HIV-positive patient with a mouse myeloma cell line. Flow cytometry was used to determine surface reactivity of the HuMAb on HIV-infected and non-infected cells. Radioimmunoprecipitation was employed to elucidate the antigen recognized by the HuMAb. A cell survival assay was used to determine the ability of the HuMAb to neutralize divergent isolates of HIV-1 in the presence or absence of complement. A gp120-CD4 inhibition enzyme-linked immunosorbent assay (ELISA) was developed in order to initiate studies to determine the mechanism of neutralization by the HuMAb. Results: An anti-HIV HuMAb was generated that neutralized two HIV-1 isolates (IIIB and MN) without complement and which neutralized one divergent isolate (RF) and one clinical isolate in the presence of complement. This HuMAb, designated S1-1, was found, by flow cytometric analysis, to react with the surface of HIV-1-infected but not with uninfected cells. Radioimmunoprecipitation analysis demonstrated that S1-1 binds to native HIV gp120, but not dithiothreitol (DTT)-treated gp120. In addition, HuMAb S1-1 did not bind to denatured HIV antigens in Western blot analysis. HuMAb S1-1 effectively inhibited the binding of gp120 to soluble CD4 in ELISA. Conclusions: These results suggest that the epitope recognized by S1-1 is conformational and conserved among diverse HIV-1 isolates and may represent an uncharacterized HIV neutralizing domain within or close to the CD4 binding domain on gp120. HuMAb S1-1 might have a role to play in vaccine development or passive immunotherapy. Keywords: Anti-HIV human monoclonal antibodies, HIV neutralization, complement-dependent neutralization of HIV.

AB - Objective: The purpose of this study was to develop and characterize human monoclonal antibodies (HuMAb) that neutralize HIV-1. Design: Based upon previous studies involving the generation of HuMAb that neutralize other enveloped viruses, we thought it feasible to generate HuMAb that might neutralize HIV-1. Methods: A HuMAb was generated by fusing splenic B-cells from an HIV-positive patient with a mouse myeloma cell line. Flow cytometry was used to determine surface reactivity of the HuMAb on HIV-infected and non-infected cells. Radioimmunoprecipitation was employed to elucidate the antigen recognized by the HuMAb. A cell survival assay was used to determine the ability of the HuMAb to neutralize divergent isolates of HIV-1 in the presence or absence of complement. A gp120-CD4 inhibition enzyme-linked immunosorbent assay (ELISA) was developed in order to initiate studies to determine the mechanism of neutralization by the HuMAb. Results: An anti-HIV HuMAb was generated that neutralized two HIV-1 isolates (IIIB and MN) without complement and which neutralized one divergent isolate (RF) and one clinical isolate in the presence of complement. This HuMAb, designated S1-1, was found, by flow cytometric analysis, to react with the surface of HIV-1-infected but not with uninfected cells. Radioimmunoprecipitation analysis demonstrated that S1-1 binds to native HIV gp120, but not dithiothreitol (DTT)-treated gp120. In addition, HuMAb S1-1 did not bind to denatured HIV antigens in Western blot analysis. HuMAb S1-1 effectively inhibited the binding of gp120 to soluble CD4 in ELISA. Conclusions: These results suggest that the epitope recognized by S1-1 is conformational and conserved among diverse HIV-1 isolates and may represent an uncharacterized HIV neutralizing domain within or close to the CD4 binding domain on gp120. HuMAb S1-1 might have a role to play in vaccine development or passive immunotherapy. Keywords: Anti-HIV human monoclonal antibodies, HIV neutralization, complement-dependent neutralization of HIV.

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