Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein

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Abstract
Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens. Respiratory syncytial virus (RSV) causes significant morbidity and mortality in children, yet an efficacious vaccine remains unavailable. Antibodies that preferentially recognize the prefusion conformation of the fusion (F) glycoprotein, particularly those that bind antigenic site Ø at the membrane-distal apex, potently neutralize infection and have aided vaccine design. Here we characterize AM14, a potent human antibody, which we show recognizes a novel epitope midway between the membrane-proximal region and the apex of the prefusion F trimer. The epitope is evenly distributed across two protomers, causing AM14 to be uniquely trimer-specific and, surprisingly, cleavage-dependent. These results indicate that the prefusion trimer is antigenically distinct from the monomer. Our findings also demonstrate that epitopes other than site Ø can be the target of extremely potent neutralizing antibodies and thus provide a new target for structure-based vaccine design. Recognition of this novel epitope could make AM14 an ideal candidate for strategies that combine passive prophylaxis with vaccination, since binding of AM14 would not block elicitation of antibodies against site Ø. Due to its unique specificity, AM14 will also be valuable for probing the conformation of RSV F-based vaccine antigens designed to be in the furin-cleaved trimeric prefusion conformation.

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