Flexible RSV prefusogenic fusion glycoprotein exposes multiple neutralizing epitopes that may collectively contribute to protective immunity

Human respiratory syncytial virus (RSV) is a significant cause of lower respiratory tract infection in infants, young children, and older adults. There is no licensed vaccine and prophylactic treatment options are limited and not widely available in developing countries with the greatest disease burden. The RSV fusion (F) glycoprotein is a primary target of host immunity and thus a major focus for vaccine development. The native F glycoprotein structure is flexible and undergoes significant rearrangements from the metastable prefusion to a stable postfusion structure with neutralizing epitopes on intermediate structures. We hypothesize trimeric vaccine strategies that recapitulate the breathable F quaternary structure, and provide accessibility of B-cells to epitopes on intermediate conformations, may collectively contribute to protective immunity, while ridge prefusion F structures restrict access to key protective epitopes. To test this hypothesis, we used the near full-length native prefusogenic F as a backbone to construct three prefusion F variants with substitutions in the hydrophobic head cavity: 1) disulfide bond double mutant (DS), 2) space filling hydrophobic amino acid substitutions (Cav1), and 3) DS plus Cav1 substitutions (DS-Cav1). In this study, we compared the immunogenicity of prefusogenic F to the immunogenicity of the prefusion F variants in two animal models. Native prefusogenic F was significantly more immunogenic producing high titer antibodies to prefusogenic, prefusion, and postfusion F structures compared to animals immunized with prefusion F DS or DS-Cav1. Importantly, native prefusogenic F elicited antibodies that targeted neutralizing epitopes including prefusion-specific site zero (O) and V as well as conformation-independent neutralizing sites II and IV. Immunization with prefusion F DS or DS-Cav1 elicited antibodies primarily targeting antigenic sites O and V with little or no detectable antibodies to other key neutralizing sites. Animals immunized with native prefusogenic F also had significantly higher neutralizing antibodies that cross-neutralized RSV A and B subtypes while immunization with DS or DS-Cav1 elicited neutralizing antibodies primarily to the A subtype. We conclude that breathable trimeric vaccines that closely mimic the native F-structure, and incorporate strategies for B-cell accessibility to protective epitopes, are important considerations for vaccine design. F structures locked in a single conformation restrict B-cell access to neutralizing epitopes that may collectively contribute to destabilizing F-trimers important for broad protection. These results also have implications for vaccine strategies targeting other type 1 integral membrane proteins.