An HIV-1 gp120 envelope human monoclonal antibody that recognizes a C1 conformational epitope mediates potent antibody-dependent cellular cytotoxicity (ADCC) activity and defines a common ADCC epitope in human HIV-1 serum

Abstract

Among nonneutralizing HIV-1 envelope antibodies (Abs), those capable of mediating antibody-dependent cellular cytotoxicity (ADCC) activity have been postulated to be important for control of HIV-1 infection. ADCC-mediating Ab must recognize HIV-1 antigens expressed on the membrane of infected cells and bind the Fcγ receptor (FcR) of the effector cell population. However, the precise targets of serum ADCC antibody are poorly characterized. The human monoclonal antibody (MAb) A32 is a nonneutralizing antibody isolated from an HIV-1 chronically infected person. We investigated the ability of MAb A32 to recognize HIV-1 envelope expressed on the surface of CD4(+) T cells infected with primary and laboratory-adapted strains of HIV-1, as well as its ability to mediate ADCC activity. The MAb A32 epitope was expressed on the surface of HIV-1-infected CD4(+) T cells earlier than the CD4-inducible (CD4i) epitope bound by MAb 17b and the gp120 carbohydrate epitope bound by MAb 2G12. Importantly, MAb A32 was a potent mediator of ADCC activity. Finally, an A32 Fab fragment blocked the majority of ADCC-mediating Ab activity in plasma of subjects chronically infected with HIV-1. These data demonstrate that the epitope defined by MAb A32 is a major target on gp120 for plasma ADCC activity.

Fig. 1.

Detection of Env expression on the cellular membrane of the A1953_SupT1 cell line. The overlaying histograms represent the staining of chronically infected A1953-SupT1 cells after incubation with no Ab(unstained), with the secondary Ab alone (black curve), and with primary and secondary Abs (green curve). The MFI of the positive population is reported for the conditions with detectable staining. The cells were stained with five different concentrations (range, 20 to 0.25 μg/ml) of the primary MAbs A32, 17b, and 2G12 and with palivizumab, which was used as a negative MAb control. The IgG preparations HIVIG and IVIG were used as positive and negative controls, respectively.

Fig. 2.

Longitudinal detection of Env expression on the cellular membrane of HIV-infected CD4⁺ T cells. For each day of culture, the histograms report the staining of activated PB CD4⁺ T cells infected with transmitted/founder primary CH077.t infectious molecular clones and the laboratory-adapted NL4-3 virus. The staining of the uninfected control is also reported. The overlaying histograms represent the staining of HIV-1-infected CD4⁺ T cells after incubation with no Ab (unstained), with the secondary Ab alone (black curve), and with primary and secondary Abs (green curve). A total of 100,000 to 200,000 CD4⁺ T cells were stained with 10 μg/ml of the MAbs A32, 17b, and 2G12 as well as with the IgG preparation HIVIG as a primary Ab source. Palivizumab and IVIG were used as negative controls for the MAb and IgG preparation, respectively. Max, maximum.

Fig. 3.

ADCC activity. gp120_WITO-coated (A), gp140_CH040-coated (B), A1953 chronically infected (C), and NL-LucR.T2A-BaL.ecto HIV-1-infected (D) CEM.NKR_CCR5 cells were used to detect the ability of MAbs A32, 17b, 2G12, b12, VRC01, and 2/11c to mediate ADCC activity. Palivizumab was used as a negative control. Each MAb was tested starting at a concentration of 40 μg/ml using 4-fold dilutions. The results are reported as percent GzB activity after the background was subtracted. The results represent the average of triplicate experiments ± standard deviation. The black dotted line represents the cutoff for positive results (5% GzB activity). PBMC from a healthy HIV-1 seronegative donor were used as a source of effector cells with an effector-to-target cell ratio of 30:1 in all experiments.

Fig. 4.

ADCC activity. Activated PB CD4⁺ T cells from a healthy HIV-1-seronegative donor after a 3-day infection with the IMC NL-LucR.T2A-BaL.ecto were used as target cells. Autologous purified NK cells were used as effector cells at effector-to-target (NK:T) ratios of 10 and 40 to 1. The MAbs A32, HIVIG (positive control), and palivizumab (negative control) were tested at the concentrations indicated on the x axis. The results are reported as the average ± standard deviation of percent GzB activity after background subtraction observed in triplicate experiments. The black dotted line represents the cutoff for positive results (5% GzB activity).

Fig. 5.

Inhibition of ADCC activity by A32 Fab fragment. The A1953_CEM.NKR_CCR5 cell line was used as target cells, and PBMC from a healthy HIV-1-seronegative donor were used as a source of effector cells. The effector-to-target cell ratio was 30:1 in all experiments. The results are reported as percent GzB activity after background was subtracted. The values above the bars represent the percentage of inhibition when A32 Fab fragment, 17b Fab fragment, 7B2 Fab fragment, and palivizumab were used in the preincubation. (A) The target cells were preincubated with medium only (no pretreatment), with 10 μg/ml of the A32 Fab fragment, or with 10 μg/ml of palivizumab. The results represent the average of results from triplicate experiments. After being washed, the cells were incubated with 10 and 1 μg/ml of MAbs A32 and 2G12. (B) The plasma collected from 14 HIV-1-infected donors was added to A1953_CEM.NKR_CCR5 target cells preincubated as previously described with medium only (no pretreatment), A32 Fab fragment, 17b Fab fragment, 7B2, or palivizumab. The MAbs A32 and 17b were used as positive controls.