Antibody Binding and Neutralization of Primary and T-Cell Line-Adapted Isolates of Human Immunodeficiency Virus Type 1

Flow cytometric analysis of MAb binding to 168P and 168C Envs. (A, C, E, and G) Neutralization sensitivity of 168P (dark line) and 168C (light line) viruses to the indicated MAbs was determined using U87-CD4-CXCR4 cells as described in Materials and Methods. Infected cells (foci) were determined by microscopic analysis of immunochemically stained monolayers, and the number of foci was compared to that obtained in the absence of MAb. (B, D, F, and H) COS-7 cells were transfected to express GFP and either 168P Env or 168C Env as described in Materials and Methods. Flow cytometric analysis of MAb binding to GFP-positive cells expressing either 168P Env (dark line) or 168C Env (light line) is illustrated. MAbs were used at the discriminating concentrations determined by neutralization as indicated in the legend to Fig. 3.

Summary of MAb binding to 168P and 168C Envs. MAb binding was determined by flow cytometry as described in Materials and Methods and quantitated using MFC computation (CELLQuest software). The respective MFC values for binding to 168P and 168C Envs were normalized relative to HIVIG binding to account for modest differences in Env expression. The normalized values for MAb binding to 168P and 168C Envs are plotted. Among all experiments, MFC values for HIVIG binding to 168P and 168C Envs were 70 ± 35 and 67 ± 28, respectively. The ratio of HIVIG MFC values within any experiment (1.07 ± 0.39) was also consistent with the ratio of transfection efficiencies (1.01 ± 0.13). Representative MAb binding values compared within one experiment are shown. Purified MAbs were used at a concentration of 10 μg/ml (except for MAbs 58.2 and 59.1, which were used as a 1:100 dilution of ascites fluids), and sCD4 was used at a concentration of 50 μg/ml. At these concentrations, the relative amounts of neutralization of 168P and 168C, respectively, are shown in parentheses (as percentages); few if any of the antibody reagents significantly neutralized 168P. Symbols:, 50.1 (0, 95); ⊕, 58.2 (55, 99); , 59.1 (20, 70); ○, 257-D (50, 97);, 268-D (50, 95); ●, 447-52D (75, 99);, 559-64D (45, 95);, IgGb12 (25, 55); ⧫, F105 (30, 65); ▴, 17b (30, 80); ▵, sCD4 plus 17b (not applicable); ■, 2F5 (60, 75); □, F240 (0, 0);, sCD4 (80, 99 at 10 μg/ml).

Summary of MAb binding to 320SI and 320SI-C3.3 Envs. Methods and MAb concentrations are as described in the legend to Fig.3. At these concentrations, the relative amounts of neutralization of 320SI and 320SI-C3.3, respectively, are shown in parentheses (as percentages); none of the antibody reagents significantly neutralized 320SI. Symbols:, 50.1 (0, 45); ⊕, 58.2 (10, 70); , 59.1 (15, 70); ⊚, 257-D (0, 60);, 268-D (0, 0); ●, 447-52D (2, 95);, 559-64D (20, 30); ◊, lgGb12 (25, 99); ⧫, F105 (10, 70); ▴, 17b (10, 60); ▵, sCD4 plus 17b (not applicable); ■, 2F5 (40, 99); □, F240 (0, 0);, sCD4 (0, 99 at 10 μg/ml). The mean ratio of HIVIG MFC values for 320SI and 320SI-C3.3 Envs was 0.87 ± 0.21, and these values varied among experiments similarly to those of 168P and 168C.

Relative capture of PI and TCLA virions. Virus stocks were prepared from supernatants of acutely infected peripheral blood mononuclear cells (PI virus) or H9 cells (TCLA virus). The virion capture ELISA was performed as described by Nyambi and colleagues (42). V3-loop-directed MAbs 58.2, 268-D, 257-D, and 447-52D were bound to microtiter wells in 100 μl at a concentration of 10 μg/ml; HIVIG was bound at 50 μg/ml. Virions were subsequently captured from 100 μl containing 100 ng of p24 per ml. The amount of virion captured by the specific MAbs is expressed as a percentage of that captured by HIVIG, in order to normalize results across virus stocks. For clarity, we show the relative capture of TCLA virion versus PI virion as the ratio of the HIVIG-normalized percentages. In the experiments shown, the underlying amounts of virion p24 captured by HIVIG were as follows: 168P, 902 pg/ml; 168C, 927 pg/ml; 320SI, 370 pg/ml; and 320SI-C3.3, 257 pg/ml. The amounts of 168P and 320SI PI virus captured by the MAbs were as follows, respectively: 58.2, 299 and 384 pg/ml; 268-D, 439 and 116 pg/ml; 257-D, 665 and 196 pg/ml; and 447-52D, 864 and 180 pg/ml. The relative capture of 168C and 168P is shown in dark bars, and 320SI-C3.3 and 320SI in light bars. As calculable, correspondingly less TCLA virion was captured than PI virion. ∗, 320SI-C3.3 virions whose V3 loop sequence contains an alteration of the nominal MAb epitope.

MAb capture of infectious PI and TCLA virions. Virions were captured from 168P and 168C virus stocks (dark and light symbols, respectively) using streptavidin-coated M-280 Dynabeads to which biotinylated MAb 50.1 had been bound. Infectivity retained by the MAb was assessed by culturing U87-CD4-CXCR4 cells with the extensively washed magnetic beads, and the numbers of infected cell foci were determined as described in Materials and Methods. Incubations of virions with MAb-coated beads were at 4 or 37°C (circle and square symbols), and retained infectivity is compared to that in the initial virus stock used for capture (triangle symbols). Streptavidin-coated beads that were incubated with nonbiotinylated MAb 50.1 (mock) served as specificity controls (data not plotted; all, ≤5 foci/well).