Human parainfluenza virus type 3 (HPIV3) is the P1-Respiroviruses prototype for advancement of vaccines and therapeutic antibodies against respiroviruses due to the advanced state of knowledge of structure and function of the viral surface proteins: HN (receptor-binding protein; hemagglutinin-neuraminidase) and F (fusion protein). Respirovirus HN and F form a functional complex on the virus surface and mediate entry by fusing the viral envelope directly with the cell membrane. Structural transitions in the HN/F complex during sequential steps of entry will be characterized using cryo-electron microscopy (cryo-EM) and tomography (cryo-ET) to identify optimal antigenic targets for respirovirus vaccines and antibodies. HPIV3 and human parainfluenza virus type 1 (HPIV1) bracket the diversity of known respiroviruses making HPIV1 ideal for testing strategies established with HPIV3. We will determine the utility of each surface glycoprotein as a target for immunity and how best to present the antigen(s) by evaluating three vaccine platforms (subunit, mRNA, rVSV). Aim 1: Define structure-function relationships of HN and F and extend the findings from prototype HPIV3 to HPIV1. Structures of HN, F, and HN/F complexes and their intermediates for the prototype respirovirus HPIV3 will be obtained to address gaps in basic understanding of the functions of the envelope proteins in viral entry and assembly, reveal essential functional interactions between the proteins, and inform immunogen design for vaccines and antibodies. Immunogenicity of proteins will be assessed in mice and iterated back to antigen design. Results will be extended to HPIV1 to assess generalizability. Aim 2: Identify monoclonal antibodies to prototype respirovirus HPIV3 HN and F with a high genetic barrier to resistance and assess the generalizability of strategies using HPIV1. We will produce HPIV3- binding mAbs from human B cells, compare their binding affinity, epitopes, and neutralization potencies; identify neutralization mechanisms, leveraging mAbs that bind one or both proteins, different domains, and different intermediate states of HN/F during entry; and evaluate mechanisms of escape. Abs that block critical entry functions of HN/F, are protective, and have a high genetic barrier to resistance will be assessed for in vivo protective/therapeutic capacity. Antigens that elicit such desirable antibodies will iteratively aid vaccine antigen design. HPIV3-based approaches will be generalized and tested against HPIV1. Aim 3: Comparison of vaccine platforms expressing HPIV3 HN, F, or HN/F complex. Immunogenicity of mRNA- and VSV-expressed vaccine candidates will be evaluated in mice and compared to subunit antigens. All three vaccine platforms (subunit, mRNA, rVSV) will be assessed for efficacy in cotton rats (HPIV3 model), with iteration back to Aims 1 and 2. Generalizability of each platform design will be determined with HPIV1. The results will define optimal immunogens and vaccine pla...