title: Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro.
abstract: OBJECTIVE
  The purpose of this study was to develop and characterize human monoclonal antibodies (HuMAb) that neutralize HIV-1.
  
  
  METHODS
  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.
authors.affiliation: Department of Internal Medicine, University of Arizona, Tucson.
authors.forename: D F
authors.initials: DF
authors.lastname: Lake
authors.forename: T
authors.initials: T
authors.lastname: Kawamura
authors.forename: T
authors.initials: T
authors.lastname: Tomiyama
authors.forename: W E
authors.initials: WE
authors.lastname: Robinson
authors.forename: Y
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Y
authors.initials: Y
authors.lastname: Masuho
authors.forename: E M
authors.initials: EM
authors.lastname: Hersh
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D005434:Flow Cytometry; D015483:HIV Antibodies / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000276:immunology / Q000378:metabolism; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D009500:Neutralization Tests; D015531:Radioimmunoprecipitation Assay; D012995:Solubility
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120