Fig. 8
Biophysical characterization of sequential binding events of bnAbs with the NFL Env trimer. The top panel illustrates binding by ITC of PGT122 and PGV19 individually and then sequentially to BG505 NFL.664 produced in HEK 293F cells to which kifunensine and swainsonine were added (resulting in oligomannose glycoforms only) and depicted within the green outlined box). The top right illustration highlights the location of PGT122 and PGV19 at the N332-supersite and the CD4bs, respectively, on one protomer as a colored cartoon. The three protomers of the Env trimer are shown in separate colors in surface representation. The second panel shows the binding of the individual antibodies and then their successive binding to BG505 NFL.664 produced in HEK293F cells (comprising of oligomannose, complex and hybrid glycans as detected by mass spectrometry studies, outlined by a pink box). The middle illustration on the right shows different glycoforms modeled on N301 (Man9: green, complex glycan: magenta) and the location of PGV19 and PGT122 as viewed from the trimer apex. The third panel describes the effect of the removal of N276 glycan on the binding of PGT122 and PGV19 individually, followed by the sequential binding of both antibodies to the N276D mutant. The bottom right illustration shows the distance between the variable regions of PGV19 and PGT122 on adjacent protomers. All values of Kd are nanomolar, enthalpy change (∆H) is cal/mol, and entropy (∆S) is cal/mol/deg. Binding stoichiometry (N) is directly affected by uncertainties in protein concentration measurement, total active molecules in the sample, and glycan heterogeneity. Associated errors/uncertainties are less than 10% of the average of at least two independent binding measurements
