Fig 1.
eCD4-IgG1 mediates potent ADCC activity.
(A) CEM.NKR-CCR5-LTR-Luc cells were infected with HIV-1 isolates 89.6, NL4-3, or YU2. Three or four days post-infection, cells were mixed at a 10:1 effector to target ratio with an NK cell line expressing human CD16a in the presence of the indicated eCD4-IgG1, eCD4-IgG2, or CD4-IgG1 concentrations. ADCC responses, defined as a loss of luciferase signal in relative light units (RLU), were measured after an 8 hour incubation. Dotted line represents 50% of maximal luciferase signal from infected targets incubated with NK cells in the absence of any inhibitor. Results are expressed as means +/- standard error of mean (S.E.M.) (n = 3). Data are representative of at least three independent experiments. (B-C) HEK293T cells were transfected to express the YU2 (B) or BG505 (C) Env with a deletion in its cytoplasmic tail (ΔCt) to increase expression on the cell surface. Cells were then incubated with the indicated concentrations of CD4-IgG1 or eCD4-IgG1, washed, and binding was measured by flow cytometry using a secondary antibody recognizing the human Fc domain. Error bars represent a range of two measurements.
Fig 2.
eCD4-Ig induces changes in antibody binding to HIV envelope glycoprotein.
(A-G) HEK293T cells were transfected to express the BG505 Env with a deletion in its cytoplasmic tail to increase expression on the cell surface. Cells were pre-incubated with varying concentrations of eCD4-Ig with mouse Fc domains (eCD4-mIg), as indicated. Cells were washed and then incubated with 0.4 μg/mL of the indicated antibodies, and analyzed by flow cytometry. Mean fluorescence intensity values (MFI) are normalized to the value of antibody binding in the absence of eCD4-Ig or CD4-Ig. Error bars represent range (n = 2). Data are representative of at least three independent experiments.
Fig 3.
eCD4-IgG2 enhances ADCC activity of V3-loop and CD4i antibodies.
An ADCC assay similar to that described in Fig 1 was used. Effector and target cells infected with HIV-1 isolates 89.6, NL4-3, or YU2 were incubated with the V3-loop antibodies 447-52D (A) and F425-B4e8 (B), the CD4i antibodies 17b (C) and A32 (D), the CD4bs antibody VRC01 (E), or 2G4, a control anti-Ebola glycoprotein antibody (F), at the indicated dilutions, either alone (open symbols) or in the presence of 1 μg/mL eCD4-IgG2 (filled symbols). ADCC activity was determined by luciferase activity after 8 hour incubation. Results are represented as means +/- S.E.M. (n = 3). Data are representative of at least three independent experiments.
Fig 4.
eCD4-IgG2 enhances ADCC activity of HIV-1 infected sera.
(A) ADCC assays similar to those described in Fig 1. CEM.NKR-CCR5-LTR-Luc target cells were infected for 3 days with YU2. Effector cells were added at a 10:1 ratio in the presence of 3-fold serial dilutions beginning at 1:32 dilution of human sera from uninfected or HIV-1 positive patients alone (red) or in the presence of 1 μg/mL eCD4-IgG2 (blue), VRC01-IgG2 (green), or 10-1074-IgG2 (purple). ADCC activity was determined by luciferase activity after an 8 hour incubation. (B) ADCC activity of eCD4-IgG1, eCD4-IgG2, VRC01-IgG2, and 10-1074-IgG2 was measured in a similar manner as in (A). Results are represented as means +/- S.E.M. (n = 3). Data are representative of at least two independent experiments. (C) 50% ADCC titers were calculated from curves in (A) as the dilution at which lines crossed the 50% maximal luciferase value. Paired t-test, ***p<0.001. (D) ADCC assay similar to those described in A. In this case, ADCC activity of serum 9121658 was tested alone (red) and in combination with 1 μg/mL eCD4-IgG1 (light blue), eCD4-IgG2 (dark blue), or CD4-IgG2 (burgundy).
Fig 5.
eCD4-IgG2 enhances ADCC activity of HIV-1 infected sera against reactivated latently infected cells.
OM-10.1 (A, C) or ACH-2 (B, D) cells lines were reactivated with TNFα (A, B) or vorinostat (SAHA) (C, D) for 24 hours prior to being mixed at a 10:1 effector to target ratio with an NK cell line expressing human CD16a in the presence of a 1:1000 dilution of serum 9121658 alone or in combination with 1 μg/mL of eCD4-IgG2, or CD4-IgG2, or VRC01-IgG2. ADCC responses, calculated based on intracellular p24 staining, were measured after a 5 hour incubation. Results are expressed as means +/- S.E.M. (n = 3). Data are representative of at least three independent experiments. Unpaired t-test, ***p<0.001, **p<0.01, *p<0.05, n.s. not significant.