Large outbreaks of RVFV can have a devastating impact on human and animal health; however, there are currently no approved vaccines for use outside the areas of endemicity in Africa. In these areas, the widespread use of available livestock vaccines has been limited due to safety concerns or poor immunogenicity. Early live attenuated constructs (i.e., Smithburn and MP12) were associated with abortion or teratogenesis in pregnant animals (8
). Inactivated VLP-like vaccines are much safer but require the use of adjuvant or multiple boosters for complete protection. Recently, our laboratory described a rationally designed, reverse genetics-derived vaccine candidate that is safe and efficacious in livestock (7
). As an additional countermeasure against RVFV, we paired the robust efficacy of this vaccine with the enhanced safety inherent in nonreplicating constructs. The resulting VRPRVF
undergo only one round of infection and are biologically confined to the initially infected cells, but they can actively synthesize viral RNA and express viral nucleoprotein and polymerase.
particles are morphologically indistinguishable from wild-type virus but lack four genes, those encoding virulence factors NSs and NSm and structural proteins Gn and Gc. Deletions of NSm (6
) and NSs (38
) have been shown to reduce virulence of RVFV in adult rodents. The NSs protein inhibits the host immune response to RVFV infection through multiple mechanisms (3
); therefore, its absence or mutation is a common feature of many RVF vaccine candidates (5
). Additional full-length deletions of genes encoding the structural proteins Gn and Gc confine VRPRVF
replication to the initially infected cells. The resulting inability to spread within the host dramatically reduces the chance of vaccine-induced pathogenicity and likely explains the safety of VRPRVF
infections in suckling mice, particularly given the rapid and uniform mortality seen with intracranial inoculation of suckling mice with RVF viruses.
Although extremely attenuated, VRPRVF
, like RVFV, contain the polymerase and nucleoprotein, the two factors required for viral replication, allowing for viral RNA expression and de novo
viral protein synthesis in the target cells. Intracellular replication of single-stranded RNA viruses (including members of the Bunyaviridae
) initiates a strong innate immune response via Toll-like receptors and/or the cytoplasmic RIG-I-like helicases, culminating in the production of important antiviral proteins, including IFN (15
). In wild-type RVFV infection, the NSs protein abolishes these host responses. However, immunization with replicating VRPRVF
lacking the NSs should allow for unobstructed production of IFN and ISGs, thus preserving critical aspects of the antiviral response. Indeed, in multiple experiments, we demonstrated a significantly stronger immune response and associated protective efficacy in VRPRVF
-immunized mice relative to those in nr-VRPRVF
- and mock-immunized mice.
Mice immunized with VRPRVF
produced significantly higher levels of total IgG and neutralizing antibodies than those in nr-VRPRVF
-immunized mice and were completely protected from the virulent virus challenge at 28 dpi, suggesting that replication is critical for robust immunity and subsequent protection. As early as 12 hpi, clear differences in host response were already apparent between the mice immunized with VRPRVF
and those immunized with nr-VRPRVF
. Relative to both nr-VRPRVF
- and mock-immunized mice, VRPRVF
immunization resulted in significant systemic induction of IFN-inducible genes, including those encoding STAT1, IRF7, ISG15, RIG-I, LPG2, and MDA5. These genes stimulate the expression of important players in the cellular defense against viruses, including PKR, OAS, IRFs, MX1, and major histocompatibility complex (MHC) classes I and II (20
). Activation of ISGs, particularly MHC, provides a mechanism for the improved antibody response and protection seen after immunization with replicating VRPRVF
. Additionally, induction of very early cell-mediated and subsequent adaptive immune responses in VRPRVF
-immunized mice was evident from the significant upregulation of CCL4 (MIP-1β) and CXCL9 (MIG) expression in the liver and CCL3 (MIP-1α) and CXCL10 (IP-10) expression in the liver and brain. These chemokines play important roles in attracting various immune cells, including monocytes/macrophages, NK cells, and T cells, and in mediating T cell activation, aiding in initiation of cell-mediated and humoral adaptive immunity.
The rapid onset of a systemic antiviral response suggested that VRPRVF immunization could confer early protection. VRPRVF were found to be highly efficacious against virulent virus challenge within days of immunization; a single dose of VRPRVF provided 60% protection by just 1 dpi and complete protection by 4 dpi. This early efficacy suggests that VRPRVF could be a valuable control measure in the field. If RVFV was introduced into an area with large naïve populations, immunization with VRPRVF early in the outbreak could prevent rapid viral spread throughout and beyond the affected region. Furthermore, the low genetic diversity and single serotype of the virus suggests that a VRPRVF vaccine would likely be broadly protective against all known strains of RVFV.
The efficacy of VRPRVF
immunization against a virulent virus challenge 100,000-fold higher than the LD50
at early and late time points was remarkable. This protection likely hinges on the ability of the VRPRVF
, administered subcutaneously and in a single dose, to elicit a robust immune response in distant tissues within hours of immunization. This systemic response to VRPRVF
inoculation is clearly illustrated by the upregulation of antiviral genes in the liver and brain after vaccination. To explain the host-wide effect of localized VRPRVF
immunization, we hypothesize that immunization results in VRPRVF
infection of resident macrophages or dendritic cells in the skin. Recent work has demonstrated that macrophages and dendritic cells are permissive to replication and are important targets of RVFV infection (28
). Given the absence of the NSs protein in the VRPRVF
construct, active replication within these cell types should stimulate a strong IFN response, as shown in vitro
), leading to a systemic antiviral response. At the time points tested in these experiments, we did not detect upregulation of the tested IFN subtypes. However, IFN must clearly have been produced within the host to induce the downstream expression of ISGs that were detected in the liver and brain. The bulk of IFN synthesis may occur at the site of immunization in locally infected macrophages or dendritic cells and then be dispersed systemically. Alternatively, IFN induction might be detectable in the liver and brain only at earlier time points or as subtypes not evaluated here. RVFV is highly sensitive to IFN, and the rapid onset of a strong IFN response associated with VRPRVF
immunization provides a plausible explanation for early protection against challenge.
Replication-competent particles are a safe vaccine approach, much like inactivated or VLP-like constructs, yet they stimulate a stronger immune response and therefore provide higher levels of protection from virulent challenge with just a single immunization. Virus-specific VRP vaccine candidates have been described for other diseases, including classical swine fever virus (35
) and Venezuelan equine encephalitis virus (22
), as well as several vaccines using an alphavirus VRP backbone (1
). A similar report of the ability of RVF replicon particles to protect against virulent challenge was very recently published (23
). Here, we show that VRPRVF
immunization rapidly and systemically initiates a strong cytokine and chemokine response with the resulting protection seen as early as 24 h postimmunization. Further, we demonstrate that the active replication that defines VRPRVF
, and distinguishes these particles from classical VLP, is critical for strong innate and adaptive immune responses and, subsequently, complete protection from challenge. Identification of initially infected cells at the site of immunization (presumably resident macrophage and/or dendritic cells of the skin) and determination of their role in the early innate response is ongoing. Further evaluation of VRPRVF
efficacy in livestock and nonhuman primates is a critical next step in proving the utility of this method, with the ultimate goal of developing a product for human use.