Dis. Our data demonstrate that both DS-Cav1 and MEDI7510 vaccination robustly elicit F-specific antibodies ENMD-2076 Tartrate and B cells, but DS-Cav1 elicited antibodies that more potently neutralized both RSV A and B. The superior potency was mediated by antibodies that bind antigenic sites on the apex of pre-F that are not present on post-F. In the memory (CD27+) B cell compartment, vaccination with DS-Cav1 or MEDI7510 elicited B cells with different epitope specificities. B cells preferentially binding the pre-F probe were activated in DS-Cav1-vaccinated participants, but not in MEDI7510-vaccinated participants. Our findings emphasize the importance of using pre-F as an immunogen in humans due to its deterministic role in eliciting highly potent neutralizing antibodies and memory B cells. One Sentence Summary: A respiratory syncytial virus prefusion subunit vaccine elicited higher potency neutralizing antibodies than a postfusion subunit vaccine in humans. Editors Summary: Function Follows Form. Respiratory syncytial virus (RSV) can cause severe infection in infants and older adults; however, no vaccine for RSV is currently approved. Candidate vaccines that target the RSV fusion (F) protein have been developed. Older vaccines use the postfusion, or Post-F, version of the protein, whereas new vaccines employ a prefusion stabilized, or Pre-F, immunogen. Here, Chang and Phung directly compared immune responses ENMD-2076 Tartrate elicited by a Post-F vaccine, MEDI7510, and a Pre-F vaccine, DS-Cav1. The authors found that DS-Cav1 elicited antibodies that more potently neutralized RSV A and B than MEDI7510. This enhanced response was mediated by the presence of antibodies that could bind sites on the F protein that were only present in the Pre-F state. Together, these data highlight the value of structure-based vaccine design. INTRODUCTION Respiratory syncytial virus (RSV) is an orthopneumovirus in the family that is responsible for millions of severe respiratory illness cases in infants, older adults, and the immunocompromised worldwide (2C8). Almost all infants are infected with RSV by the age of two, with repeated infections throughout life (9). Despite the ubiquitous nature of RSV infection and magnitude of its impact, no licensed vaccine is currently available, indicating a major gap in global health. Clinical evaluation of a formalin-inactivated whole virus RSV vaccine adjuvanted with aluminum salts (FI-RSV) in infant cohorts during the 1960s resulted in enhanced ENMD-2076 Tartrate respiratory disease in vaccinated infants, hampering the momentum of RSV vaccine development for several decades (10, 11). The majority of subsequent RSV vaccines have been based on the fusion (F) protein, an attractive target due to its high degree of antigenic and genetic conservation (11). The clinical success of palivizumab, an anti-F monoclonal antibody, illustrates the potential for F-directed, neutralizing antibodies to prevent severe disease. Subsequent studies have also demonstrated that a high concentration of neutralizing activity in the serum correlates with protection (12C18). The F glycoprotein exists in two major conformations on the surface of RSV: the active conformation, prefusion F (pre-F), and the inactive conformation, postfusion F (post-F). Pre-F displays six major antigenic sites (?, I, II, III, IV, V), whereas only four of these sites (I, II, III, IV) are retained following transition to post-F (11). Prior to the stabilization of pre-F, many F-based vaccine candidates in clinical testing used post-F as the vaccine antigen, given its high stability; however no post-F vaccine candidates have advanced to licensure (19, 20). The stabilization and structural characterization of Hdac8 pre-F led to the identification of highly neutralization-sensitive epitopes (sites ? and V) exclusive to the pre-F surface, motivating efforts to use pre-F ENMD-2076 Tartrate as a vaccine antigen capable of eliciting high titers of neutralizing antibody (11, 12, 21C26). A pre-F-exclusive monoclonal antibody targeting site ?, nirsevimab (MEDI8897), has also demonstrated efficacy in preterm, late preterm, and term infants (27, 28). Although both conformations of F have been represented as vaccine candidates in the pipeline for clinical evaluation, direct comparison of immunity elicited by pre- and post-F vaccines has not been done in humans. Serum samples and peripheral blood mononuclear cells (PBMCs) were collected from a phase 1 clinical trial evaluating a pre-F (DS-Cav1) vaccine antigen as well as two independent phase 1 and 2 clinical trials evaluating a post-F vaccine antigen (MEDI7510). DS-Cav1 is a stabilized pre-F vaccine, recently evaluated in phase 1 clinical testing (NCT03049488), and administered to healthy adult volunteers (ages 18 to 50) in 50.