PROJECT SUMMARY/ABSTRACT Rotavirus (RV) is the leading cause of diarrhea-associated morbidity and mortality in children younger than five worldwide. While RV vaccines have substantially decreased RV deaths, children in low- and middle- income countries remain at risk of life-threatening RV disease because of significantly lower vaccine effectiveness compared to high-income countries. We recently identified a potential role for Enterovirus B (EV- B) infection in reducing oral RV vaccine (ORV) performance. Specifically, EV-B infection at the time of vaccination is associated with a lack of seroconversion to ORV in a cohort of infants from Ghana. However, the signaling pathways and mechanisms associated with this interference have not yet been defined. We hypothesize that EV-Bs induce epithelial-derived antiviral cytokines to limit both ORV replication and anti-RV immune responses. Determination of the mechanisms underlying EV-B-mediated interference with ORV will leverage complementary analyses in ex vivo human intestinal organoids, in vivo mouse models, and non-invasive fecal analyses from a human clinical trial cohort of infants receiving RV vaccines. Representative EV-B strains consistent with those identified in the Ghanaian infant cohort and which are prevalent in countries with reduced vaccine efficacy have been selected for these studies. We will interrogate the innate antiviral signaling pathways induced by EV-B infection, as well as test whether they are required to limit ORV or RV replication, in both pediatric human organoids, including isogenic lines where key innate immune genes are knocked-out, and wild- type, transgenic or knock-out mouse lines. EV-B co-infection effects on development of adaptive immune responses to RV will also be evaluated in murine models. Additionally, fecal samples from an ongoing clinical trial evaluating ORV and non-replicating RV vaccine clinical efficacy will be used to validate EV-B interference with ORV, define EV-B-associated antiviral cytokines, and assess whether EV-B also interferes with non- replicating RV vaccines. We have assembled a team of investigators with extensive expertise in the experimental RV systems, clinical vaccinology, and virome analyses required to define the molecular mechanisms underlying the important and persistent clinical challenge of low ORV performance in low- and middle-income countries. Completion of this proposal will provide key insights into EV-B-mediated interference with ORV, as well as broadly into virus-virus interactions in the intestinal environment and their consequences for development of immune responses. By identifying key factors that regulate vaccine responsiveness, we aim to facilitate development and testing of evidence-based interventions to improve RV vaccine performance.