Characterization of single-cycle flavivirus particles for use as a vaccine to prevent West Nile disease and to examine immune responses to flavivirus infection
Douglas Gregory Widman
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West Nile virus (WNV) is responsible for the largest outbreak of viral encephalitis in the history of North America, yet there are no vaccines available to prevent this disease. To address these needs we have developed RepliVAX WN, a single-cycle flavivirus (SCFV)-based vaccine to prevent West Nile disease. RepliVAX WN contains a C-deleted WNV genome, and is produced in trans-complementing cell lines that express WNV C. When used for vaccination, RepliVAX WN infects a single cell where the genome replicates and drives the production of highly antigenic subviral particles (SVPs) and NS1 without producing infectious virions. Thus, RepliVAX is expected to be highly potent yet exhibit a safety profile superior to traditional live-attenuated viral vaccines.\r\nHere we demonstrate that RepliVAX WN can be safely passaged in C-expressing cell lines and that this blind passage selected for mutations used to engineer a second-generation RepliVAX WN with an enhanced in vitro growth phenotype. When evaluated in mouse and hamster models of WN disease, this second-generation RepliVAX was safe, exhibited 100% protective efficacy, and induced significantly higher antibody levels than the parental virus. Furthermore, we observed that RepliVAX WN-induced antibody levels remain steadily at high levels for at least 6 months after vaccination of hamsters, and all animals were protected from lethal WNV challenge at this time. Evaluation in non-human primates indicated that one or two doses of RepliVAX WN was safe, induced WNV-specific antibody responses, and protected animals from WNV viremia. \r\nHaving demonstrated the usefulness of RepliVAX WN as a vaccine to prevent WN disease, we were interested in the immunological mechanisms underlying vaccine immunity. We observed that although RepliVAX WN vaccination induces high levels of interferon (IFN) alpha, the ability to respond to either type-I or type-II IFNs was not required for the development of activated B cells, IgG, IgM, or neutralizing antibody titers. Type-I IFN signaling did, however, play a role in viral gene expression, as in vivo imaging of animals inoculated with luciferase-expressing SCFVs revealed 1000-fold greater bioluminescence in the absence of a type-I IFN response. The affect of this IFN response on gene expression was dramatic, but short lived and did not appear to play a role in SCFV persistence, as SCFV gene expression was detectable for at least 18 days after SCFV inoculation. Taken together these results demonstrate the usefulness of SCFVs like RepliVAX WN as vaccines to prevent flavivirus disease, and tools with which to examine immune responses to viral infection.