Molecular basis for protection and cross-protection by human antibodies targeting the parainfluenza virus hemagglutinin-neuraminidase protein

Human parainfluenza viruses (PIVs) are a leading cause of respiratory illness, particularly in vulnerable populations where infection can lead to severe disease. Despite their clinical impact, there are currently no licensed vaccines or effective antiviral treatments available. PIVs have two large surface proteins, the fusion and hemagglutinin-neuraminidase (HN) proteins, both of which are targets of neutralizing antibodies. In this study, we identified and characterized two human monoclonal antibodies (mAbs), 5217-2 and 5217-9, which bind recombinant PIV3 HN protein, bind PIV3-infected cells, and are neutralizing in vitro. We determined the binding epitopes of the PIV3 HN-specific mAbs via biolayer interferometry and found mAb 5217-9 targets a previously defined neutralizing epitope while mAb 5217-2 binds a unique epitope, enabling a more complete understanding of the antigenic landscape. To further understand the newly defined epitope, we determined a cryo-electron microscopy (cryo-EM) structure of mAb 5217-2, which revealed an epitope adjacent to the PIV3 HN protein active site. We also determined the structure of the previously discovered anti-PIV3 HN mAb PIV3HN-09, which was previously shown to be partially protective in vivo. In a hamster challenge model of PIV3, mAb 5217-2 was determined to significantly reduce lung viral titers, demonstrating its protective capacity. Furthermore, as the site 2-directed mAb PIV3HN-05 was previously shown to cross-neutralize PIV1, we evaluated its protective efficacy in an animal challenge model with PIV1, which demonstrated a reduction in lung viral titers. Overall, these findings provide new insights into the antigenic epitopes on the PIV3 HN protein to support structure-based vaccine design efforts and demonstrate new protective mAbs for both PIV3 and PIV1.