Antibodies of the PGT121 family compete with sCD4, but not PGV04, for binding to gp120 in solution and on the cell-surface.

<p>A. Fold change in binding affinity (K_d) for sCD4 and PGV04 interactions with gp120 when antibodies of the PGT121 family are first pre-complexed with gp120. The order from left to right is as on the inset legend from top to bottom. Negative values indicate a decrease in binding affinity of sCD4 or PGV04 in the presence of PGT121 antibodies, positive values represent an increase in binding affinity and a value of ∼1 represents no effective change in binding affinity. Sequential ITC binding experiments show that sCD4 has over 100 to 650 fold decrease in binding affinity to gp120 when antibodies of the PGT121 family are pre-complexed with gp120. On the other hand, pre-complexing PGT121 antibodies with gp120 does not lead to a change in binding affinity for PGV04 to gp120. These results suggest that antibodies of the PGT121 family do not sterically block access to the CD4 binding site (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s008" target="_blank">Fig. S8A</a>), but disturb CD4 binding, possibly by interfering with gp120 conformational changes associated with CD4 binding. No sCD4 competition by PGT121 antibodies is observed for a core-miniV3 gp120 construct, indicating that modulatory elements of gp120 such as C1, V1/V2 and fully length V3 are important in modulating sCD4 competition. B. SEC-purified complexes of gp120 with various Fabs were tested for their ability to bind CD4+ TZM-bl cells by FACS. 17b+gp120 (black) and 2G12+gp120 (orange) complexes bound well to CD4+ TZM-bl cells. On the other hand, a PGT123+gp120 complex (red) was not able to engage CD4+ TZM-bl cells. Lack of binding of the PGT123+gp120 complex to CD4+ TZM-bl cells is comparable to similar lack of binding of gp120 in complex with the CD4-binding site antibody PGV04 (blue). A similar inability to bind CD4+ TZM-bl cells was observed for gp120 in complex with PGT121 and PGT122 antibodies (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s009" target="_blank">Fig. S9</a>). PE-A represents the relative intensity of detected Fab on the surface of CD4+ TZM-bl cells. Curves with filled areas indicate Fab alone (negative control), whereas curves with hollow areas are for the Fab-gp120 complexes. C) sCD4 and D) PGV04 binding to cells expressing JRFL Env on their surface as observed by flow cytometry. PGT123 Fab (pink), b12 Fab (yellow) and 17b Fab (green) were pre-incubated with the cells in titrating amounts at 37°C before being exposed to a constant amount of either C) sCD4 or D) PGV04. As expected, b12 Fab that targets the CD4 binding site directly competes with CD4 binding, and 17b Fab, which binds to the co-receptor binding site, enhances CD4 binding. PGT123 Fab competes with sCD4 binding to the same extent as b12 Fab. A similar level of sCD4 competition was observed for PGT121 and PGT122 antibodies (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s009" target="_blank">Fig. S9</a>). On the other hand, PGT123 Fab does not compete with PGV04, a CD4 binding site targeted antibody that does not induce conformational changes upon binding <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342-Falkowska1" target="_blank">[36]</a>. Binding curves are represented by plotting the dimensionless mean fluorescence intensity (MFI) of C) sCD4 and D) PGV04 binding as a function of Fab concentration.