UCSF

Abstract Neisseria gonorrhoeae, the causative agent of gonorrhea, represents an escalating global health concern because of the rapid spread of antimicrobial resistance and the continued lack of an effective vaccine. The lipooligosaccharide (LOS) epitope targeted by monoclonal antibody 2C7 is highly conserved and expressed by the majority of clinical isolates, highlighting its potential as an attractive target for therapeutic and vaccine-based interventions. To define the molecular basis for 2C7 recognition, we structurally characterized mAb 2C7 bound to an LOS-derived octasaccharide. NMR and a 1.6 Å crystal structure of the Fab 2C7–octasaccharide complex showed that mAb 2C7 recognizes the β- and γ-glycan chains through an extensive network of hydrogen-bonding and hydrophobic interactions within the Fab binding cleft. MD simulations indicated that β and γ chains form stable, specific interactions with 2C7, whereas α-chain contacts are transient. Guided by structure, we synthesized a heptose II-linked β/γ-chain tetrasaccharide with its carrier-protein conjugate bound mAb 2C7 by ELISA and Western blotting. ITC revealed affinities comparable to the octasaccharide, confirming retention of key binding determinants. Binding was enthalpy-driven, consistent with crystallographic and MD-defined polar contacts. NMR confirmed engagement of all four residues, established as the minimal 2C7 epitope. These results provide a structural framework for the rational development of glycan-based vaccines and antibody therapeutics targeting antimicrobial-resistant N. gonorrhoeae.