Generation and characterization of neutralizing human monoclonal antibodies against human immunodeficiency virus type 1 Tat antigen

Abstract

The human immunodeficiency virus Tat regulatory protein is essential for virus replication and pathogenesis. From human peripheral blood mononuclear cells of three Tat toxoid-immunized volunteers, we isolated five Tat-specific human monoclonal antibodies (HMAbs): two full-length immunoglobulin G (IgG) antibodies and three single-chain fragment-variable (scFv) antibodies. The two IgGs were mapped to distinct epitopes within the basic region of Tat, and the three scFvs were mapped to the N-terminal domain of Tat. The three scFvs were highly reactive with recombinant Tat in Western blotting or immunoprecipitation, but results were in contrast to those for the two IgGs, which are sensitive to a particular folding of the protein. In transactivation assays, scFvs were able to inhibit both active recombinant Tat and native Tat secreted by a transfected CEM cell line while IgGs neutralized only native Tat. These HMAbs were able to reduce viral p24 production in human immunodeficiency virus type 1 strain IIIB chronically infected cell lines in a dose-dependent manner.

FIG. 1.

Immunoreactivity of HMAbs to rTat. (A) rTat submitted to SDS-PAGE and Western blotting was detected by antiserum from a Tat-immunized human volunteer (lane 1), scFv G1 (lane 2), scFv G2 (lane 3), scFv J1 (lane 4), IgG J3B2 (lane 5), IgG B1E3 (lane 6), and control IgG (lane 7). (B) rTat was first immunoprecipitated with the antibodies indicated for panel A, SDS-PAGE and Western blotting were carried out, and immunodetection was performed using antiserum from a Tat-immunized rabbit.

FIG. 2.

Neutralization of Tat transactivation by HMAbs. LTR-dependent CAT production by HL3T1 cells was measured after preincubation of active rTat with increasing concentrations of antibodies (A) or after coculture with CEM cells transiently expressing native Tat 1-72 in the presence of fixed amounts of antibodies (5 μg/ml for G1 and 10 μg/ml for B1E3, J3B2, and control antibody) (B). Results are expressed as percentages of transactivation, considering 100% as the value obtained with Tat alone. Data provided are the mean values from three independent experiments (standard deviation, <10% in all experiments).

FIG. 3.

Inhibition of HIV-1 IIIB replication by HMAbs. Production of p24 HIV antigen by H9 chronically infected cells in the presence of HMAbs or control antibody was measured after 3 days of culture. Results are expressed as percentages of p24 production, considering 100% as the value obtained without antibodies in the culture medium. Data provided come from a typical experiment.