The impact of RVFV on both livestock and human health is extensive and strongly epidemiologically linked, making RVFV uniquely suitable for a one-health prevention approach through animal vaccination programs. Unfortunately, since the development of the original inactivated and live-attenuated Smithburn (or Smithburn-related) serially passaged vaccines in the 1940s and 1950s, the acceptance of RVFV vaccines for livestock use has been low except in emergency situations. Significant barriers to routine and consistent use of these vaccines have included high costs of manufacture, a poorly defined genetic identity, poor efficacy, no capacity to differentiate vaccinated from naturally infected livestock, and, most importantly, the inherent risk of vaccination in pregnant animals due to associated teratogenesis and abortion (9
). These factors and others have created a strong reluctance among herdsmen and national governments to implement the sustained vaccination efforts necessary to prevent the massive periodic animal losses and human deaths associated with outbreaks of RVF in areas of endemicity.
To address these concerns, we developed a rationally designed vaccine candidate, ΔNSs-ΔNSm rZH501, based on the complete deletion of the 2 known RVFV virulence factors, the NSs and NSm genes. We have previously reported on the safety of this vaccine in rodents (4
) and the potential to discriminate vaccinated from naturally infected animals using a 2-way DIVA ELISA technique based on the virus NP and NSs proteins (26
). Here, we extend that work to the first comprehensive examination of the safety and efficacy of this novel RVFV vaccine in healthy nonpregnant and pregnant sheep.
The vaccine caused no p.v. fever, inappetance, or impact on clinical health and was immunogenic at dosages ranging from 1.0 × 103
to 1.0 × 105
PFU in healthy nonpregnant sheep ( to ). A large-scale follow-up study in timed-pregnant animals demonstrated that the vaccine was safe for both dam and fetus even when given early in gestation (day 42), when the risk of RVFV vaccine-related teratogenesis is highest ( and ) (10
). Each pregnant animal that received the vaccine delivered at least one healthy lamb free of any clinically apparent physical or neurological defects. Lamb tissues were negative for residual vaccine genome and antigen and free of any vaccine-related histological abnormalities (). The vaccine was also highly immunogenic in pregnant ewes, causing a robust increase in IgM and IgG titers by 4 to 8 days p.v. (A and B).
The vaccine conferred protection from viremia, pyrexia, clinical disease, and abortion in all animals challenged intravenously with 1.0 × 106 PFU of virulent rRVFV ( and data not shown). In marked contrast, sham-vaccinated animals developed rapid viremia, pyrexia, and inappetance, and all aborted fetuses within 6 to 12 days p.c. (). Molecular and histological testing of the aborted fetal materials confirmed that the virulent challenge virus was the proximate cause of the observed fetal death and abortion (B, top row, and data not shown).
Furthermore, we detected no spread of the vaccine between vaccinated and nonvaccinated animals even when housed under highly confined laboratory conditions. This lack of vaccine virus spread was likely attributable to the absence of p.v. viremia (). This suggests minimal risk of transmission and dissemination of the vaccine into the environment by blood-feeding vectors, such as mosquitoes. Thus, we conclusively demonstrated that the ΔNSs-ΔNSm rRVFV vaccine can provide a safe and efficacious tool against RVFV in a relevant livestock host, including pregnant animals.
The ΔNSs-ΔNSm rRVFV vaccine was designed to overcome some of the safety limitations of previous serially passaged (Smithburn or MP-12) or naturally occurring mutant (clone 13) vaccines by completely removing the entire open reading frame of the 2 main RVFV virulence factors encoded on two different virus RNA genome segments. In contrast to the MP-12 vaccine strain, which contains 9 amino acid substitutions accumulated over multiple passages in cell culture, the ΔNSs-ΔNSm rRVFV vaccine lacks a total of 264 amino acids (aa) (normally encoded by the S RNA segment) and 129 aa (normally encoded by the M RNA segment), which renders this vaccine much less likely to revert to virulence due to random nucleotide substitutions introduced by viral polymerase errors.
Reassortment of genome RNA segments by members of the family Bunyaviridae
during coinfection has been reported in laboratory-derived and field-isolated viruses (2
). Importantly, the absence of p.v. viremia or dissemination in ΔNSs-ΔNSm rRVFV-vaccinated animals renders reassortment of cocirculating vaccine and wild-type viruses extremely unlikely. However, even if such a single reassortment event were to occur with the ΔNSs-ΔNSm rRVFV vaccine, the complete deletion of 2 virulence genes on two different RNA segments would ensure that any putative reassortant or recombinant strain was attenuated, as it would still contain at least one major genomic lesion. This is a significant theoretical safety advantage compared to the clone 13 strain vaccine, which carries only an ~70% deletion of the NSs gene (16
An essential step toward the adoption of an RVFV vaccine for use in areas of nonendemicity is the capacity to differentiate infected animals from vaccinated animals. We show here the utility of a novel 3-way DIVA ELISA (NP, NSs, and NSm) as a platform to provide rapid (~2.5-h), inexpensive, and high-throughput screening of sheep and other livestock without the need for reagents prepared in a high-containment laboratory. This assay showed that, as expected, all vaccinated animals generated antibodies only to the NP structural proteins and none to the deleted NSs and NSm proteins (D and data not shown). Interestingly, we observed variations in the levels of anti-NSs and -NSm antibodies in control virulent-virus-infected animals. In these animals, the titers against the NSs and NSm proteins ranged from 100 to 400 by 37 days p.c. (D). To our knowledge, this is the first time that the immunoreactivity of the NSm protein has been quantitated in any species. From our data, it is clear that a 3-way DIVA assay would likely be more reliable than our originally described 2-way assay (anti-NSs/NP only) in confirming vaccinated or infected status based on the presence or absence of anti-NSs, anti-NSm, and anti-NP antibodies. Further testing and validation of this new diagnostic platform and adaptation to a field-ready rapid diagnostic tool is under way using a large array of livestock and wildlife specimens collected from areas of endemicity in Africa and Saudi Arabia.
The immunity induced by the ΔNSs-ΔNSm rRVFV vaccine was striking. The body temperature profiles, clinical health, qRT-PCR, and serological assays demonstrated that a robust and sterilizing immune response was generated in the 9 vaccinated animals challenged with 1.0 × 106 PFU of virulent RVFV. In these animals, infection with a potentially lethal dose of RVFV was accompanied only by a marked increase in antibody titers (B and D). The 3-way DIVA ELISA clearly showed that this rise was directed specifically to the structural proteins of the inoculated virus, not to the nonstructural NSs and NSm proteins. This finding is significant, since NSs and NSm proteins could only be expressed following virulent-virus infection and replication. Coupled with the lack of detectable p.c. viremia or fever, this finding strongly suggests that a sterilizing immune response was generated by the ΔNSs-ΔNSm rRVFV vaccine.
Control programs for RVFV provide an ideal test case for the merits of a one-health approach to zoonosis prevention. A DIVA-compatible vaccine platform would be clearly beneficial to livestock growers by directly improving animal health and economically by allowing the control of outbreaks while respecting international trade regulations requiring serological proof of disease-free herd status. Our data presented here demonstrate that the ΔNSs-ΔNSm rRVFV vaccine could be utilized to fulfill these needs as a safe and effective tool to combat the significant impacts of RVFV on humans and animals.