A VSV-based oral rabies vaccine was sentineled by Peyer's patches and induced a timely and durable immune response.

The global eradication of canine-mediated human rabies remains an ongoing public health priority. While conventional oral rabies vaccines (ORVs) have demonstrated partial success in interrupting zoonotic transmission, current formulations necessitate improvements in both immunogenic profiles and mechanistic clarity. Herein, we present a recombinant vesicular stomatitis virus (VSV)-vectored vaccine candidate (rVSVΔG-ERA-G) engineered to express the glycoprotein of the rabies virus (RABV) ERA strain, substituting the native VSV glycoprotein. Preclinical evaluation across multiple mammalian species (Mus musculus, Canis lupus familiaris, Felis catus, Vulpes lagopus, and Nyctereutes procyonoides) revealed rapid seroconversion and sustained neutralizing antibody responses. Challenge experiments demonstrated 100% survival efficacy in pre-exposure prophylaxis models, with partial protection observed in post-exposure scenarios. Safety assessments confirmed significant attenuation of neurotropism and absence of horizontal transmission or environmental shedding. Furthermore, evidence showed that rVSVΔG-ERA-G is recognized by Peyer's patches (PPs), where a cascade activation of immune cells occurred. From another perspective, the absence of functional microfold cells in PPs hampered the initiation and progression of immune responses. This proof-of-concept study establishes rVSVΔG-ERA-G as an ORV candidate with enhanced biosafety and cross-species immunogenicity. The elucidation of M cell-dependent mucosal priming mechanisms provides a rational framework for optimizing the targeted delivery of ORVs.