Rift Valley fever virus (RVFV), a mosquito-borne Phlebovirus, causes significant economic losses and health risks in humans and livestock, primarily ruminants. The virus leads to abortions in pregnant animals and mortality in newborns. Current RVF control relies on vaccination, with both inactivated and live-attenuated vaccines in use. Inactivated vaccines require multiple administrations, while live-attenuated vaccines, such as the Smithburn and Clone 13 strains, although effective with a single dose, exhibit residual virulence, limiting their use in young and pregnant animals. The development of safer and more effective live-attenuated vaccines is crucial. This study focuses on a novel four-segmented RVFV (RVFV-4s) vaccine, which was previously shown to be safe in mice, lambs, and pregnant ewes. This four-segmented approach, involving splitting the M segment encoding glycoproteins Gn and Gc, and the absence of the major virulence factor NSs, contributes to the vaccine's safety profile. The study aims to further evaluate the safety and efficacy of vRVFV-4s in young ruminants, adhering to OIE and European Pharmacopoeia guidelines.

Existing RVF vaccines have limitations. Inactivated vaccines, while safe, require multiple doses for optimal efficacy. Live-attenuated vaccines, such as the Smithburn strain and Clone 13, offer single-dose effectiveness but carry risks of residual virulence, particularly in young or pregnant animals. Clone 13, despite its safety improvements by lacking a significant portion of the NSs gene, shows vertical transmission and teratogenic effects at high doses. Reverse genetics approaches offer a pathway to develop safer and more effective live-attenuated RVF vaccines by introducing attenuating mutations or deletions. Previous research by the authors demonstrated the safety of a four-segmented RVFV (RVFV-4s) in young lambs and pregnant ewes. This new approach, based on splitting the M segment into two and eliminating the NSs gene, resulted in a significantly attenuated virus. This study builds on these findings to further assess the safety and efficacy of vRVFV-4s in a broader range of ruminants and challenge conditions.

The study comprised three main parts: a dissemination, shedding, and spread (DSS) study; a reversion to virulence (RTV) study; and efficacy studies in lambs, goats, and calves. The DSS study involved vaccinating lambs with a high dose of vRVFV-4s and co-housing them with sentinel lambs to monitor viral dissemination, shedding, and spread. Viral RNA was measured in plasma, organs, and swabs. The RTV study assessed the potential for vRVFV-4s to revert to virulence through serial passages in lambs. Organ suspensions from the initial vaccinated lambs were used to inoculate subsequent groups. Efficacy studies involved vaccinating lambs, goat kids, and calves with vRVFV-4s at different doses and challenging them with homologous (strain 35/74) and heterologous (strain ZH501) RVFV strains. Clinical signs, viremia (viral RNA and virus isolation in plasma), viral RNA in organs (liver and spleen), and antibody responses (neutralization test and anti-N ELISA) were monitored. Detailed procedures, including animal husbandry, cell culture, virus propagation, RNA isolation and RT-qPCR, virus isolation, serological assays, and statistical analysis are described in the Methods section. Humane endpoints (HEP) were defined and strictly adhered to. Ethical approvals were obtained for all animal procedures.

The DSS study showed that vRVFV-4s did not disseminate, shed, or spread to sentinel animals. Transient low levels of viral RNA were detected in the plasma of vaccinated lambs for only 1–2 days post-vaccination. The RTV study demonstrated that vRVFV-4s did not revert to virulence after serial passage in lambs. Efficacy studies showed that a single vaccination with vRVFV-4s provided complete protection against homologous RVFV challenge in lambs, goats, and calves. No vaccinated animals developed fever or viremia, and viral RNA was absent in their organs. Homologous challenge in mock-vaccinated animals resulted in fever, viremia, and viral RNA detection in organs, with lambs showing the most severe disease. Significant differences in viremia and antibody responses were observed between species, with goats exhibiting the highest neutralizing antibody titers. Finally, a single vRVFV-4s vaccination protected young lambs against a heterologous RVFV challenge (strain ZH501), demonstrating broad protection against genetically distinct RVFV strains.

The results confirm the high safety profile of the vRVFV-4s vaccine and its effectiveness in inducing protective immunity in young ruminants. The absence of viral dissemination, shedding, and reversion to virulence highlights the vaccine's safety. The consistent protection across different ruminant species against both homologous and heterologous RVFV strains suggests that vRVFV-4s could be a valuable tool for controlling RVF outbreaks. Interspecies differences observed in viremia and antibody responses emphasize the importance of species-specific considerations in vaccine development and deployment. The transient presence of low viral RNA levels in the vaccinated animals suggests limited replication of the vaccine virus, which explains its safety profile.

This study demonstrates that the vRVFV-4s vaccine is safe and highly effective in protecting young sheep, goats, and calves against RVFV infection. The vaccine's inability to disseminate, its lack of reversion to virulence, and its ability to provide broad protection against different RVFV strains make it a promising candidate for RVF control. Future research could focus on optimizing the vaccine dose for different species and evaluating its efficacy in the field.

While the study showed promising results, some limitations exist. The study was conducted under controlled laboratory conditions, which might not fully reflect real-world conditions. The sample sizes, especially in some of the subgroup analyses, could be considered relatively small. Further field trials are needed to validate the findings and evaluate the vaccine's long-term efficacy and safety under field conditions. Additionally, the study primarily focused on the humoral immune response; future studies investigating cell-mediated immunity would provide a more comprehensive understanding of the vaccine's mechanism of action.