Assessment of Single‐Cycle M‐Protein Mutated Vesicular Stomatitis Virus as a Safe and Immunogenic Mucosal Vaccine Platform for SARS‐CoV‐2 Immunogen Delivery

The goal of the next-generation COVID-19 vaccine is to provide rapid respiratory tract protection with a single dose. Circulating antibodies do not protect the olfactory mucosa from viral infection, necessitating localized mucosal immunization. Live attenuated vesicular stomatitis virus (VSV_MT)-based COVID-19 vaccines effectively stimulate mucosal immunity in animals, though safety concerns remain, particularly in immunocompromised populations. A viral vector capable of single-cycle replication may face less stringent regulatory requirements. A replication-defective VSV_MT is developed with its G protein replaced by a SARS-CoV-2 spike protein (S) mutant, where residues K986 and V987 are substituted by prolines (S2P). This studies show that single-cycle VSV_MT encoding Omicron subvariant S2P (VSV_MT-S2P) is safe in both healthy and immunocompromised animals treated with cyclophosphamide (CP). Significant antibody and T-cell responses against the spike protein are observed in VSV_MT-S2P vaccinated healthy animals. Intramuscular VSV_MT-S2P administration induces neutralizing antibody responses comparable to those from replication-competent VSV_MT-S. In immunocompromised animals, lower and delayed immune responses are observed. Thus, single-cycle M-protein mutated VSV offers a safe and effective platform for SARS-CoV-2 immunogen delivery. Remarkably, replication-competent VSV_MT-S caused no pathogenicity and elicited potent mucosal immunity via intranasal administration, highlighting its potential as a mucosal COVID-19 vaccine.