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The Coronavirus Disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of systemic and mucosal IgA and T cell responses, completely prevents SARS-CoV-2 infection in the upper and lower respiratory tracts, and likely confers sterilizing immunity in most animals. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission, and curtailing pandemic spread. ### Competing Interest Statement M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and on the Scientific Advisory Board of Moderna. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. M.J.H. is a member of the DSMB for AstroZeneca and founder of NuPeak Therapeutics. The Baric laboratory has received unrelated funding support in sponsored research agreements with Takeda, Pfizer, and Eli Lily.

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