Fig. 5. Antibodies produced p.i. by 10E8-UCA^H B cells face competition from wild-type antibodies.

a, Orientation of mature 10E8 Fab in relation to the MPER peptide in the published Protein Data Bank (PDB) 4G6F crystal structure¹² (heavy chain, white; light chain, dark gray; MPER peptide, purple; top left) and surface rendering and positioning of the critical YxFW residues of HCDR3 (bottom left), representative GT10.2-10E8-UCA^H mAb in complex with a glycan-knockout (KO) version of 10E8-GT10.2 (heavy chain, yellow; light chain, dark gray; targeted MPER graft with GT mutations, purple; top center; structure aligned and oriented to the MPER peptide as in the top left) and designed binding pocket (green) and engagement of the GT10.2-10E8-UCA^H mAb HCDR3 (yellow) compared to mature 10E8 HCDR3 (white; bottom center) and representative GT10.2-WT mAb in complex with 10E8-GT10.2 (heavy chain, red; light chain, dark gray; targeted MPER graft with GT mutations, purple; top right) and binding pocket (green; bottom right). b, Overlay of the GT10.2-10E8-UCA^H and GT10.2-WT mAbs described in a, showing the representative competitor response in relation to on-target response (top) and overlay of the GT10.2-WT mAb with mature 10E8 mAb from a (bottom). Arrows indicate clashes of the antibodies. c, Representative flow cytometry histograms showing the mean fluorescence intensity of fluorescently labeled GT10.2-10E8-UCA^H-PE mAb to 10E8-GT10.2-coated streptavidin beads in the presence of GT10.2-10E8-UCA^H mAb or GT10.2-WT mAb or in the absence of other mAbs. Data are derived from one of two experiments. d,e, Representative flow cytometry plots (d) and quantifications (e) of GC B cells and CD45.2⁺ B cells in GC B cells in the spleen at day 7 p.i. in CD45.1 wild-type mice adoptively transferred with CD45.2 10E8-UCA^H B cells to reach a frequency of 1:10⁴ CD45.2 10E8-UCA^H B cells 1 day before i.p. injection with or without 10 µg of GT10.2-WT mAb, followed by immunization with 5 µg of 10E8-GT10.2 12mer with alhydrogel 12 h later. Error bars indicate mean ± s.d. from mice in each group (n = 5). Data were analyzed with a two-tailed Mann–Whitney test and are derived from one experiment.