ABSTRACT In this R21 application, we propose to develop a universal, intranasal, parainfluenza virus 5 (PIV5)-based respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) bivalent vaccine. RSV and HMPV are two of the leading causes of acute respiratory infections (ARIs) in children, immunocompromised individuals, and the elderly. Illness ranges from asymptomatic infection to severe bronchiolitis and pneumonia, with 90-100% of children infected with RSV by 2 years of age, and HMPV between the ages of 5-10 years old. No licensed RSV or HMPV vaccine is available and there is an unmet medical need to develop safe and effective vaccines for both diseases. A recent proof-of-principle study has shown that it is possible to create a chimeric F protein (RHMS-1) by combining immunodominant epitopes from RSV F and HMPV F that retains antigenicity for both viruses, the purified protein is immunogenic and protective against RSV and HMPV challenge in mice. However, this purified protein requires an adjuvant and a multi-dose approach, the cell-mediated immune response was not studied, the duration of the antibody response has not been determined, and there is a potential safety concern for children due to the high dose of protein required for the vaccine to be immunogenic. PIV5 is a safe delivery vector for intranasal immunization. A PIV5-vectored RSV candidate vaccine (BLB-201) has already been cleared by the FDA for a phase I clinical trial this year (NCT05281263). Due to the similarities between RSV and HMPV F proteins, disease manifestation, and target populations, a bivalent vaccine is desirable to protect against ARI diseases caused by both viruses. Here, we propose to introduce the RHMS-1 novel sequence into the PIV5- vectored vaccine platform. We will also make a modification to the RHMS-1 sequence to improve F protein expression and its immunogenicity. The constructs to be evaluated include: 1) RHMS-1 pre-fusion; and 2) RHMS-1 pre-fusion form with the trimerization domain replaced with the transmembrane domain and cytoplasmic tail from the PIV5 F protein. The candidate vaccine viruses will be compared for their replication and antigen expression in vitro, and immunogenicity and protective efficacy against RSV and HMPV challenge infection in vivo. The novelty of the vaccine proposed in this R21 application relates to: 1) the use of a chimeric RSV+HMPV F protein against two pathogens (bivalent vaccine); 2) a needle-free intranasal delivery method in a safe, highly immunogenic viral vector; 3) ease of administration; and 4) the ability to induce cellular and antibody responses including mucosal immunity, which is necessary for protecting against respiratory pathogens. The project is very promising in generating an effective bivalent vaccine that could provide protection against the two leading causes of acute lower respiratory tract infections in children and older adults.