Abstract Among the common strategies to design next-generation COVID-19 vaccines is broadening the antigenic repertoire thereby aiming to increase efficacy against emerging variants of concern (VoC). This study describes a new Orf virus-based vector (ORFV) platform to design a multiantigenic vaccine targeting SARS-CoV-2 spike and nucleocapsid antigens. Vaccine candidates were engineered, either expressing spike protein (ORFV-S) alone or co-expressing nucleocapsid protein (ORFV-S/N). Mono- and multiantigenic vaccines elicited comparable levels of spike-specific antibodies and virus neutralization in mice. Results from a SARS-CoV-2 challenge model in hamsters suggest cross-protective properties of the multiantigenic vaccine against VoC, indicating improved viral clearance with ORFV-S/N, as compared to equal doses of ORFV-S. In a nonhuman primate challenge model, vaccination with the ORFV-S/N vaccine resulted in long-term protection against SARS-CoV-2 infection. These results demonstrate the potential of the ORFV platform for prophylactic vaccination and represent a preclinical development program supporting first-in-man studies with the multiantigenic ORFV vaccine.
