Fig. 4: Scanning the antigen surface through SHM and low affinity in Public B cell Lineage 1 (Lin1).
From: Engaging an HIV vaccine target through the acquisition of low B cell affinity
A sHsL, gHgL and inferred intermediate sequences within B cell Lineage 1 (Lin1_mut1-4). Lin 1 is public clonal lineage that also uses a public pattern of SHM that is conserved across vaccine recipients (Supplementary Figs. 7, 12). B sHsL, gHgL and intermediate BCR sequences were expressed as IgM BCRs in a B cell reporter system78 and evaluated by flow cytometry for binding to fluorescent versions of WT Env (red lines) vs D368R Env (blue lines). Gray histograms represent binding to BCR isotype control or binding to surface BCR negative for LC expression. Data presented represents one experiment. C sHsL, gHgL and Lin1_mut1-4 expressed as Fabs and then evaluated for binding to Env vs Env-D368R using bilayer interferometry (BLI). The equilibrium dissociation constant (KD) values were calculated by applying a 1:1 binding isotherm using vendor-supplied software (see also Supplementary Table 1). KD values above 100 µM are beyond the limit of detection for this instrument17. Data presented represents one experiment. D Permissiveness in B cell affinity selection as a window for scanning the antigen surface through SHM. Acquisition of D368R sensitivity in the membrane BCR format (see B) is underscored by lower affinity for cognate antigen, as measured in the Fab format (see C). B, C represent independent experiments to compare BCR recognition by two orthogonal methods (binding by membrane presented BCR versus monomeric antibody affinity). A phylogenetic tree denoting the positions of sHsL, gHgL and intermediates is presented in Supplementary Fig. 12.
