Structural characterization of Fab-dimerized glycan-reactive antibodies that neutralize HIV-1 A glycan shield covers the HIV-1 envelope (Env) limiting antibody access to broadly neutralizing antibody (bnAb) epitopes. 2G12 had for long been the only example of a HIV-1 bnAb that interacts solely with the Env glycan shield. Its unique VH domain-swapped architecture, with two Fab arms swapped to create a Fab-dimerized IgG, allows 2G12 to simultaneously interact with 4 glycans, thus bolstering typically weak protein-glycan binding through avidity. We have recently characterized structurally diverse Fab-dimerized, glycan-reactive (FDG) antibodies that target the HIV-1 Env glycan shield. Unlike 2G12, these newly identified FDG antibodies are not domain-swapped; instead, Fab dimerization occurred by mechanisms including inter- Fab disulfide linkage, hydrophobic and hydrogen bond interactions. We further showed that the HIV-1 Env- targeting FDG antibodies recognized a glycan cluster in the S2 subunit of the SARS-CoV-2 spike. While our results reveal diverse ways antibodies can Fab dimerize to recognize glycans clusters, several questions remain regarding the mechanisms of Fab dimerization and glycan recognition. Understanding these will provide insights into the development of B cell responses to glycans. The overall goals of this study are to understand structural determinants of antibody Fab dimerization leading to high affinity glycan recognition. That 2G12 and other FDG bnAbs specifically recognize a conserved glycan cluster on HIV-1 Envs that consists of self-sugars in a unique non-self presentation provides basis for immunological discrimination between glycans on host and invading pathogens. The scientific premise of this grant is that defining structural mechanisms for glycan recognition by Fab dimerized antibodies will allow specific targeting of diverse glycosylated pathogens. The innovations in this grant derive from (i) an expanded repertoire of FDG antibodies, (ii) the demonstration that FDG antibodies are prevalent, (iii) the finding that domain-swapped VH conformation is not necessary for HIV-1 neutralization.

In this grant we will structurally characterize HIV-1 glycan shield targeting Fab-dimerized glycan reactive (FDG) antibodies. The overall goals of this study are to define atomic level structures of novel FDG antibodies in complex with their HIV-1 Env epitopes, to elucidate the structural determinants of FDG antibody evolution, and to determine the structural basis for FDG antibody recognition of diverse Coronavirus (CoV) spikes.