Figure 4.

Epitope locations of anti-gp160 antibodies affect their abilities to redirect anti-DNP CAR-NK cells against subtype B gp160⁺ cells. (A) Illustration of the epitope locations of 12 anti-gp160 antibodies tested in this study. The image was generated based on the structures of HIV-1 BG505 SOSIP.664 Env trimer (PDB ID: 5T3Z) and MPER region (PDB ID: 6E8W).^{42, 43} (B) IFN-γ production by anti-DNP CAR-NK cells against subtype B gp160-expressing cells in the presence of different DNP-conjugated bNAbs (2 nM). IFN-γ concentrations were determined by ELISA. Data are presented as mean ± SD. Statistical significance is calculated by one-way ANOVA and Dunnett’s post-hoc test compared with the isotype control. * p<0.05, ** p<0.01, **** p<0.0001. (C) Cytotoxicity of anti-DNP CAR-NK cells against subtype B gp160⁺ cells at multiple E:T ratios and with different DNP-conjugated bNAbs (2 nM). Cells were stained with a viability dye and analyzed by flow cytometry. The percentage of cytotoxicity was calculated as [(A-B)/Ax100], in which A and B were the numbers of viable gp160⁺ cells after the cell co-culture was incubated without and with the addition of DNP-conjugated antibodies, respectively. Data are presented as the mean ± SD of triplicate samples. Statistical significance is calculated by two-way ANOVA and Tukey’s post-hoc analysis. * p<0.05,** p<0.01,**** p<0.0001 vs. the isotype control. ^## p<0.01 comparing 3BNC117 to VRC01 at the 25:1 E:T ratio. (D) Analysis of the binding potencies of bNAbs against subtype B gp160⁺ cells. Cells were stained by bNAbs or the isotype control at a series of three-fold dilutions (from 0.045 nM to 300 nM) followed by a PE-conjugated anti-human IgG. The average fluorescence intensity per cell was measured by flow cytometry. Data are presented as the mean ± SD of triplicate samples. Nonlinear regression is used to fit saturation binding curves.