The introduction of three single nucleotide mutations into the genome of the virulent RVFV ZH548 strain allows the rescue of a fully attenuated virus in mice (ZH548-rA2). These mutations are located in the viral genes encoding the RdRp and the non-structural protein NSs. This paper shows the results obtained after subcutaneous inoculation of ZH548-rA2 in adult sheep and subsequent challenge with the parental virus (ZH548-rC1). Inoculation with ZH548-rA2 virus caused no detectable clinical or pathological effect in sheep, whereas inoculation of the parental rC1 virus caused lesions compatible with viral infection characterized by the presence of scattered hepatic necrosis. Viral infection was confirmed by immunohistochemistry, with hepatocytes within the necrotic foci appearing as the main cells immunolabelled against viral antigen. Furthermore, inoculation of sheep with rA2 virus prevented the liver damage expected after rC1 virus inoculation, suggesting a protective efficacy in sheep which correlated with the induction of both humoral and cell mediated immune responses.

The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we report the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin converting enzyme 2 (K18-hACE2) mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the levels of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine