Table 2. Restoration of the sensitive phenotype in broadly resistant HIV-1 strains through site-directed mutagenesis.
| Neutralizing activity | Binding activity | Receptor usage | ||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| VRC01 class | non-VRC01 classes | CD4-related | VRC01 class | non-VRC01 classes | Cf2Th | Cf2Th | ||||||||||||||||||
| Virus strains and mutants | VRC01 | 3BNC117 | N6 | CH103 | 8ANC131 | VRC13 | VRC16 | ibalizumab | CD4-Ig | VRC01 | 3BNC117 | N6 | VRC13 | CD4.CCR5 | CCR5 | |||||||||
| IC50 | IC80 | IC50 | IC80 | IC50 | IC80 | IC50 | IC80 | IC50 | IC80 | IC50 | IC80 | IC50 | IC80 | IC50 | IC80 | IC50 | IC80 | GMFI | GMFI | RLU | RLU | |||
| CNE6 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 1.19 | >50 | >50 | >50 | 0.19 | 0.99 | 5.41 | 14.36 | 10.41±2.96 | 11.66±2.74 | 11.29±2.62 | 16.43±1.72 | 2215341 | 27 |
| CNE6_E97K | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 5.26 | >50 | >50 | >50 | 0.23 | 2.10 | 3.29 | 10.14 | 10.29±2.99 | 12.26±3.41 | 10.73±2.28 | 15.73±1.37 | 5684407 | 36 |
| CNE6_I279D | 1.10 | 15.57 | 28.15 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.04 | 0.67 | >50 | >50 | 0.05 | 0.14 | 40.90 | >50 | 13.53±3.25 | 12.78±3.23 | 11.76±2.94 | 17.43±1.78 | 1523709 | 27 |
| CNE6_H281A | 40.83 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 1.38 | 16.54 | >50 | >50 | 0.02 | 0.06 | >50 | >50 | 10.45±3.01 | 10.87±2.71 | 11.53±2.78 | 16.50±2.66 | 431281 | 13 |
| CNE6_E97K_I279D | 1.98 | 4.82 | 5.99 | 23.50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.33 | 1.83 | >50 | >50 | 0.09 | 0.23 | 29.91 | >50 | 15.10±3.54 | 15.23±3.71 | 11.66±2.55 | 17.50±2.83 | 2943819 | 10 |
| CNE6_E97K_H281A | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 9.43 | >50 | >50 | >50 | 0.04 | 0.07 | >50 | >50 | 11.00±3.46 | 11.35±3.15 | 12.66±3.75 | 15.83±1.88 | 113754 | 15 |
| CNE6_I279D_H281A | 28.72 | >50 | 9.50 | >50 | 0.51 | 2.91 | >50 | >50 | >50 | >50 | 0.11 | 1.13 | >50 | >50 | 0.58 | 2.30 | 3.04 | 10.11 | 14.01±3.86 | 15.17±3.31 | 18.75±2.45 | 16.93±2.55 | 3942741 | 32 |
| CNE6_E97K_I279D_H281A | 12.18 | 26.50 | 1.25 | 4.20 | 0.45 | 2.16 | >50 | >50 | >50 | >50 | 0.67 | 2.20 | >50 | >50 | 3.12 | >50 | 1.56 | 4.23 | 14.94±4.51 | 17.47±4.13 | 19.90±4.10 | 17.73±3.10 | 3237033 | 32 |
| CNE23 | 41.87 | >50 | >50 | >50 | 16.94 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.02 | 0.06 | >50 | >50 | 10.67±2.00 | 12.67±0.82 | 12.70±0.50 | 14.45±2.05 | 1199356 | 24 |
| CNE23_N461A | 8.34 | 24.83 | >50 | >50 | 3.19 | 14.78 | >50 | >50 | >50 | >50 | 2.55 | >50 | >50 | >50 | 0.04 | >50 | >50 | >50 | 12.13±0.92 | 13.60±1.57 | 16.34±2.24 | 14.85±0.15 | 1662466 | 51 |
| CNE23_E471G | >50 | >50 | >50 | >50 | 28.74 | >50 | >50 | >50 | >50 | >50 | 4.02 | >50 | >50 | >50 | 0.03 | 0.08 | 11.65 | 34.87 | 10.86±1.32 | 13.37±0.69 | 12.61±0.39 | 15.85±1.55 | 2482645 | 59 |
| CNE23_N461A_E471G | 8.27 | 33.34 | 13.76 | 41.92 | 6.36 | 20.02 | >50 | >50 | >50 | >50 | 2.25 | 31.52 | >50 | >50 | 0.10 | >50 | 6.05 | 43.48 | 12.60±1.55 | 14.87±1.35 | 16.26±1.16 | 16.35±1.05 | 2669833 | 39 |
| CNE63 | >50 | >50 | >50 | >50 | 16.50 | >50 | >50 | >50 | >50 | >50 | 6.74 | 44.19 | >50 | >50 | 0.01 | 0.03 | >50 | >50 | 9.18±1.17 | 9.98±0.84 | 10.35±0.80 | 15.73±1.58 | 87966 | 32 |
| CNE63_E97K | >50 | >50 | >50 | >50 | 8.85 | 46.91 | >50 | >50 | >50 | >50 | 5.90 | 35.14 | >50 | >50 | 0.01 | 0.04 | 13.15 | >50 | 9.44±1.27 | 10.31±1.27 | 10.60±0.78 | 15.60±1.36 | 85643 | 23 |
| CNE63_L281A | 22.98 | >50 | 21.62 | >50 | 4.73 | 29.35 | >50 | >50 | >50 | >50 | 1.38 | 13.78 | >50 | >50 | 0.01 | 0.04 | 21.28 | >50 | 9.84±1.11 | 10.89±1.09 | 10.62±0.76 | 15.67±1.93 | 49487 | 19 |
| CNE63_N461A | 24.27 | >50 | >50 | >50 | 1.15 | 6.24 | >50 | >50 | >50 | >50 | 4.09 | 18.65 | >50 | >50 | 0.02 | 0.17 | 9.97 | >50 | 10.16±1.11 | 10.46±0.96 | 10.99±1.28 | 14.47±1.52 | 123704 | 15 |
| CNE63_E97K_L281A | 11.70 | >50 | 13.85 | 44.62 | 2.49 | 24.11 | >50 | >50 | >50 | >50 | 0.81 | 7.63 | >50 | >50 | 0.01 | 0.03 | 10.24 | >50 | 9.69±1.31 | 10.62±1.23 | 10.83±0.52 | 14.03±1.01 | 271723 | 18 |
| CNE63_E97K_N461A | 0.03 | 17.40 | >50 | >50 | 0.86 | 3.72 | >50 | >50 | >50 | >50 | 1.96 | 19.37 | >50 | >50 | 0.02 | 0.90 | 4.81 | >50 | 10.19±0.95 | 10.32±0.82 | 11.01±1.23 | 14.30±1.82 | 200466 | 97 |
| CNE63_L281A_N461A | 3.03 | 15.79 | 6.95 | 31.89 | 2.01 | 6.38 | >50 | >50 | >50 | >50 | 0.38 | 4.18 | >50 | >50 | 0.02 | 0.35 | 8.50 | >50 | 10.42±0.96 | 11.30±0.83 | 10.54±1.30 | 14.90±1.71 | 49046 | 24 |
| CNE63_E97K_L281A_N461A | 2.21 | 5.62 | 0.30 | 3.52 | 0.38 | 1.19 | >50 | >50 | 33.48 | >50 | 0.12 | 1.33 | 1.35 | 6.68 | 0.11 | 26.63 | 1.10 | 3.22 | 12.03±0.63 | 12.93±1.52 | 13.63±1.43 | 15.73±1.01 | 293879 | 21 |
| CNE64 | >50 | >50 | >50 | >50 | 5.82 | 16.49 | >50 | >50 | >50 | >50 | 0.02 | 0.46 | >50 | >50 | 0.08 | 0.47 | 3.11 | 17.59 | 9.54±1.31 | 9.99±1.17 | 11.07±0.05 | 13.83±1.16 | 24957 | 64 |
| CNE64_E97K | >50 | >50 | >50 | >50 | 5.62 | 11.70 | >50 | >50 | >50 | >50 | 0.15 | 3.59 | >50 | >50 | 0.17 | 0.44 | 3.26 | 8.00 | 9.85±1.32 | 10.46±1.36 | 13.03±1.74 | 13.97±1.67 | 76618 | 45 |
| CNE64_L281A | 14.52 | 36.67 | 6.23 | 31.10 | 4.56 | 8.47 | >50 | >50 | >50 | >50 | 0.08 | 1.07 | 45.95 | >50 | 0.04 | 0.10 | 12.50 | 30.24 | 10.12±1.37 | 11.10±1.31 | 12.43±1.18 | 14.63±1.51 | 227521 | 64 |
| CNE64_N461A | >50 | >50 | >50 | >50 | 0.85 | 2.24 | >50 | >50 | >50 | >50 | 0.10 | 1.32 | >50 | >50 | 3.47 | >50 | 3.22 | 7.22 | 11.15±1.78 | 10.61±1.71 | 14.57±1.79 | 15.13±2.70 | 66595 | 47 |
| CNE64_E97K_L281A | 3.47 | 33.68 | 2.20 | 14.11 | 3.63 | 8.60 | >50 | >50 | >50 | >50 | 0.20 | 3.60 | 37.16 | >50 | 0.06 | 0.16 | 1.54 | 6.36 | 9.15±1.15 | 10.19±1.41 | 12.33±0.47 | 14.35±3.25 | 447707 | 56 |
| CNE64_E97K_N461A | 10.97 | 31.34 | >50 | >50 | 0.38 | 1.75 | >50 | >50 | >50 | >50 | 0.43 | 4.87 | >50 | >50 | 0.20 | 28.56 | 0.56 | 1.55 | 11.10±1.45 | 10.65±1.65 | 15.20±2.83 | 14.27±1.75 | 122324 | 34 |
| CNE64_L281A_N461A | 1.76 | 6.60 | >50 | >50 | 0.48 | 1.30 | >50 | >50 | >50 | >50 | 0.15 | 1.88 | 1.28 | 5.67 | 0.48 | 44.64 | 5.68 | 9.54 | 11.30±0.93 | 10.79±1.19 | 15.07±3.63 | 13.97±1.36 | 352783 | 36 |
| CNE64_E97K_L281A_N461A | 3.33 | 9.85 | 2.38 | 13.38 | 2.59 | 5.88 | >50 | >50 | >50 | >50 | 2.10 | 39.06 | >50 | >50 | 0.13 | 2.02 | >50 | >50 | 11.37±0.73 | 12.38±2.15 | 17.60±3.11 | 13.47±1.05 | 227625 | 27 |
| CNE66 | >50 | >50 | >50 | >50 | 0.06 | 0.25 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.05 | 0.38 | 22.75 | >50 | 8.22±0.96 | 9.49±0.05 | 13.55±0.05 | 10.75±0.55 | 9162 | 16 |
| CNE66_G281A | >50 | >50 | >50 | >50 | 0.08 | 0.22 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.14 | 1.08 | 2.08 | >50 | 7.95±0.64 | 9.22±0.40 | 13.75±0.75 | 11.05±0.85 | 522912 | 82 |
| CNE66_D459G | 16.90 | >50 | >50 | >50 | 0.03 | 0.22 | 2.12 | 16.61 | >50 | >50 | >50 | >50 | >50 | >50 | 0.04 | 0.85 | 1.74 | >50 | 8.52±1.12 | 9.33±0.11 | 12.30±0.20 | 10.80±0.50 | 88661 | 24 |
| CNE66_E97K_G281A | 9.32 | >50 | >50 | >50 | 0.02 | 0.09 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.05 | 0.48 | 1.16 | 42.74 | 8.03±0.60 | 9.28±0.45 | 13.60±1.30 | 10.68±0.93 | 44540 | 18 |
| CNE66_E97K_D459G | >50 | >50 | >50 | >50 | 0.09 | 0.38 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.12 | 0.36 | 2.38 | 18.06 | 8.37±0.84 | 9.50±0.01 | 11.50±0.70 | 10.32±0.69 | 59181 | 16 |
| CNE66_G281A_D459G | 2.89 | 7.22 | >50 | >50 | 0.08 | 0.27 | 5.39 | 36.82 | >50 | >50 | 42.85 | >50 | >50 | >50 | 0.05 | 1.78 | 1.48 | 10.92 | 9.99±0.51 | 9.74±0.04 | 12.85±0.05 | 11.15±0.45 | 48345 | 18 |
| CNE66_D459G_Q471G | >50 | >50 | >50 | >50 | 0.07 | 0.18 | 9.25 | >50 | >50 | >50 | 0.34 | 6.41 | >50 | >50 | 0.14 | 0.31 | >50 | >50 | 8.97±1.44 | 12.15±1.05 | 15.80±1.80 | 12.85±1.05 | 23815 | 21 |
| CNE66_E97K_G281A_D459G | 1.18 | 16.36 | >50 | >50 | 0.02 | 0.12 | 2.40 | 25.32 | >50 | >50 | >50 | >50 | >50 | >50 | 0.08 | 1.65 | 0.69 | 4.65 | 9.62±0.98 | 10.08±0.73 | 14.70±1.60 | 11.40±0.40 | 62833 | 18 |
| CNE66_E97K_D459G_Q471G | 2.80 | 41.18 | >50 | >50 | 0.13 | 0.25 | >50 | >50 | >50 | >50 | 6.36 | 29.98 | >50 | >50 | 0.02 | 0.10 | 5.22 | >50 | 9.27±1.43 | 11.70±1.30 | 15.10±2.40 | 12.35±0.75 | 19326 | 22 |
| CNE66_E97K_G281A_D459G_Q471G | 0.58 | 4.17 | 0.23 | 3.97 | 0.07 | 0.36 | 5.97 | 20.48 | 1.54 | 13.15 | 5.97 | 19.87 | >50 | >50 | 0.07 | 0.19 | 3.32 | 36.55 | 10.77±0.84 | 13.15±1.75 | 15.25±1.75 | 13.10±1.30 | 6224 | 22 |
| BJOX2000 | >50 | >50 | >50 | >50 | 0.81 | 2.38 | >50 | >50 | 18.13 | >50 | >50 | >50 | >50 | >50 | 0.09 | >50 | 31.84 | >50 | 11.40±0.50 | 11.30±0.30 | 14.65±0.05 | 11.70±0.40 | 87876 | 36 |
| BJOX2000_E455T | 10.08 | 25.44 | 9.17 | 19.07 | 1.37 | 5.79 | >50 | >50 | 34.29 | >50 | 48.12 | >50 | >50 | >0 | 0.04 | >50 | 13.90 | >50 | 12.40±0.40 | 13.15±0.35 | 14.75±0.05 | 12.00±0.10 | 217493 | 39 |
| BJOX2000_P461N | >50 | >50 | >50 | >50 | 0.39 | 3.07 | 0.64 | 24.57 | >50 | >50 | >50 | >50 | >50 | >50 | 0.06 | >50 | >50 | >50 | 11.75±0.05 | 11.65±0.05 | 15.55±0.75 | 11.60±0.60 | 419246 | 61 |
| BJOX2000_E455T_P461N | 2.29 | 11.90 | 1.80 | 10.81 | 0.73 | 3.81 | 19.49 | >50 | >50 | >50 | >50 | >50 | 24.63 | >50 | 0.03 | >50 | 5.27 | >50 | 10.75±0.45 | 11.70±0.50 | 13.40±0.60 | 12.60±0.60 | 2120789 | 5583 |
| BL01 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.08 | 8.85 | 2.10 | 14.26 | ND | ND | ND | ND | 537355 | 26 |
| BL01_E97K | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.15 | 19.70 | 2.25 | 8.29 | ND | ND | ND | ND | 555891 | 31 |
| BL01_Q279D | 42.98 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.10 | 0.39 | 5.64 | 44.80 | ND | ND | ND | ND | 34957 | 31 |
| BL01_E282K | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.14 | >50 | 2.06 | 12.78 | ND | ND | ND | ND | 208063 | 51 |
| BL01_I471G | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.05 | 0.34 | 12.11 | 39.53 | ND | ND | ND | ND | 421749 | 26 |
| BL01_E97K_Q279D | 11.39 | >50 | >50 | >50 | 2.70 | 10.98 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.09 | 3.08 | 1.79 | 7.97 | ND | ND | ND | ND | 115835 | 22 |
| BL01_E97K_E282K | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.04 | >50 | 0.69 | 11.72 | ND | ND | ND | ND | 469409 | 78 |
| BL01_E97K_I471G | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.07 | 2.09 | 1.86 | 14.94 | ND | ND | ND | ND | 199279 | 87 |
| BL01_Q279D_E282K | 40.68 | >50 | >50 | >50 | 2.39 | 12.20 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.06 | >50 | 2.30 | 9.14 | ND | ND | ND | ND | 470740 | 27 |
| BL01_Q279D_I471G | 31.02 | >50 | >50 | >50 | 0.67 | 19.48 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.07 | 0.15 | 28.43 | >50 | ND | ND | ND | ND | 136232 | 34 |
| BL01_E282K_I471G | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.04 | 0.59 | 8.26 | >50 | ND | ND | ND | ND | 354475 | 26 |
| BL01_E97K_Q279D_E282K | 19.86 | >50 | >50 | >50 | 3.86 | 12.97 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.17 | >50 | 0.95 | 4.38 | ND | ND | ND | ND | 413664 | 21 |
| BL01_E97K_Q279D_I471G | 25.49 | >50 | >50 | >50 | 0.36 | 1.70 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.09 | 1.08 | 16.30 | 43.57 | ND | ND | ND | ND | 197560 | 51 |
| BL01_E97K_E282K_I471G | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.17 | >50 | 3.09 | 20.72 | ND | ND | ND | ND | 724553 | 44 |
| BL01_Q279D_E282K_I471G | 31.62 | >50 | 0.52 | 9.83 | 1.94 | 28.51 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.13 | 16.92 | 6.06 | 28.75 | ND | ND | ND | ND | 253740 | 34 |
| BL01_E97K_Q279D_E282K_I471G | 23.33 | >50 | 0.79 | 6.75 | 1.85 | 7.33 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.07 | >50 | 2.71 | 14.22 | ND | ND | ND | ND | 737429 | 31 |
| TV1.29 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.06 | 0.87 | >50 | >50 | 0.02 | 0.10 | 0.56 | 2.72 | ND | ND | ND | ND | 118590 | 44 |
| TV1.29_E279D | >50 | >50 | 18.80 | 45.08 | 5.50 | 15.50 | >50 | >50 | >50 | >50 | 5.26 | >50 | >50 | >50 | 0.03 | 0.08 | 0.60 | 5.32 | ND | ND | ND | ND | 268497 | 70 |
| TV1.29_T281A | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.03 | 0.09 | >50 | >50 | 0.01 | 0.09 | 6.97 | 19.27 | ND | ND | ND | ND | 12582 | 34 |
| TV1.29_N461A | >50 | >50 | >50 | >50 | 0.12 | 16.50 | >50 | >50 | >50 | >50 | 0.81 | 2.96 | >50 | >50 | >50 | >50 | 0.71 | 1.56 | ND | ND | ND | ND | 87462 | 34 |
| TV1.29_E279D_T281A | >50 | >50 | 20.20 | >50 | 0.05 | 4.72 | >50 | >50 | >50 | >50 | 0.09 | 1.12 | >50 | >50 | 0.04 | 0.09 | 2.13 | 13.23 | ND | ND | ND | ND | 206570 | 36 |
| TV1.29_E279D_N461A | 0.74 | 10.29 | 0.90 | 6.83 | 0.09 | 1.12 | >50 | >50 | >50 | >50 | 0.87 | 4.17 | >50 | >50 | 0.16 | >50 | 1.29 | 5.38 | ND | ND | ND | ND | 372688 | 27 |
| TV1.29_E279D_T281A_N461A | 1.37 | 5.29 | 2.75 | 24.18 | 0.07 | 0.91 | >50 | >50 | >50 | >50 | 0.98 | 3.93 | >50 | >50 | 37.38 | >50 | 5.82 | 24.63 | ND | ND | ND | ND | 117690 | 31 |
| TZA125.17 | >50 | >50 | >50 | >50 | 0.23 | 2.30 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.03 | 0.61 | >50 | >50 | ND | ND | ND | ND | 28008 | 34 |
| TZA125.17_N461A | 25.80 | >50 | >50 | >50 | 0.47 | 5.51 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | >50 | 0.73 | 3.46 | 17.16 | >50 | ND | ND | ND | ND | 67566 | 34 |
| Meidian IC50/IC80(μg/ml) | <0.1 | 0.1–1 | 1–10 | 10–50 | >50 | n-fold of GMFI | 0.5–1.2 | >1.2 | RLU | <103 | 103−104 | 104−105 | 105−106 | 106−107 | ||||||||||
The numbers under “Neutralizing activity” represent the estimated mean values of IC50 and IC80 derived from at least three independent experiments. The color scheme is the same as in Table 1. The numbers under “Binding activity” represent the means and variation of estimated GMFI values from at least three independent experiments. Those that increased more than 1.2-fold relative to the original clones are indicated in blue. The numbers under ‘Receptor usage’ represent the luciferase activity in relative light units (RLU). The color scheme represents various ranges of RLU indicated below. ND: not detected.