Glycan modifications to the gp120 immunogens used in the RV144 vaccine trial improve binding to broadly neutralizing antibodies
Fig 5
Screen of MN glycan mutant supernatants for improvements to bN-mAb binding.
A FIA was used to identify 293 GnTI- expressed MN-rgp120 glycan variants exhibiting improved bN-mAb binding profiles as compared to the wildtype MN sequence expressed in GnTI- (MN358). Recombinant gp120s were expressed in GnTI- 293 cells via transient transfection, and transfection supernatants were normalized to contain ~2μg/mL. MN-rgp120 variants were captured onto 96 well black plates using a 1μg/mL concentration of mouse monoclonal antibody to an N-terminal gD tag. Binding curves to the VRCO1 bN-mAb, which binds a conformation dependent epitope in the CD4 binding site, were used to assay for maintenance of overall secondary and tertiary structure. All screening assays were performed in duplicate. MN-rgp120 glycan variants were assayed for improved antigenicity to a panel of glycan dependent bN-mAbs to be considered for further analysis.