Project summary: First identified in California in 2012, acute flaccid myelitis in children, associated with enterovirus D68 (EV-D68) infection, has been increasing in incidence with outbreaks detected every 2 years. Enteroviruses are well-known causes of central nervous system pathologies, ranging from aseptic meningitis to sometimes fatal brainstem encephalitis and myelitis, which can lead to permanent debilitating paralysis. Additionally, EV-D68 infects the respiratory tract, causing severe respiratory disease and facilitating person-to-person transmission via respiratory droplets. Despite EV-D68’s emergence as a major cause of severe respiratory and neurological disease, there are no vaccines or therapeutics available to combat and control the spread of this pathogen. HDT Bio has developed a self-amplifying replicon RNA (repRNA) vaccine platform delivered by a Lipid InOrganic Nanoparticle (LION) scheduled to enter phase I clinical trials in the first quarter of 2021 as a vaccine against COVID-19. These activities will enable rapid translation of other vaccine candidates, utilizing the same platform, into the clinic. Additionally, HDT has an ongoing program to develop broad-spectrum anti-EV-D68 antibody therapeutics, in which we have identified promising RNA-based vaccines that encode the necessary genes for production of divergent EV-D68 virus like particles (VLPs) in vivo upon intramuscular administration. Our preliminary data establishes that 1) we can launch VLPs of non-enveloped viruses from our repRNA platform, 2) we can rapidly adapt this approach for genotypic and/or antigenic variants of EV-D68, and 3) these antigens are very immunogenic in small and large animals, generating robust neutralizing antibody responses after a single dose. In this application, we propose to screen six vaccine candidates, which are currently being evaluated as a mixture in alpacas for antibody discovery efforts, to identify a single candidate that induces the best cross- neutralizing antibody responses. We will then characterize safety, immunogenicity and efficacy in neurological- and respiratory-disease mouse models of EV-D68 infection. Finally, we will evaluate safety and immunogenicity in pregnant mouse models and efficacy in birthed pups while characterizing maternal antibody transfer.