Broad and durable protection against SARS-CoV-2 and SARS-CoV by an intranasal chimpanzee adenovirus vaccine expressing tandem RBDs and nucleocapsid.

Current intramuscular COVID-19 vaccines reduce severe disease but offer limited protection against infection, enabling viral persistence, immune escape, and transmission. We developed intranasal rare-serotype chimpanzee adenovirus 68 (AdC68)-based vaccines expressing heterologous tandem receptor-binding domains from SARS-CoV-2, SARS-CoV, and MERS-CoV, fused to the SARS-CoV-2 nucleocapsid (AdC68-4RBD(XBB.1.5)-N) to enhance both B and T cell responses. Antigen integrity was confirmed by receptor binding and recognition by multiple conformation-sensitive monoclonal antibodies. In mice, intranasal AdC68-4RBD(XBB.1.5)-N elicited potent and durable mucosal and systemic immunity, including serum/saliva IgA and broad neutralizing antibodies persisting up to 40 weeks, alongside tissue-localized B and T cells. Monoclonal antibodies from long-lived bone marrow antibody-secreting cells demonstrated broad and strain-specific neutralization, providing mechanistic insight into the breadth and longevity of the antibody response. In Syrian hamsters, intranasal immunization protected against SARS-CoV-2 XBB.1.5 replication in nasal turbinates and lungs and blocked transmission for up to four months post-vaccination. Robust protection against SARS-CoV challenge was demonstrated in K18-hACE2 transgenic mice, further confirming its broad efficacy. These findings support AdC68-4RBD(XBB.1.5)-N as a promising mucosal vaccine candidate to prevent infection and transmission of pathogenic coronaviruses.