Using the single-cycle flavivirus particle replivax to study flavivirus replication and immune response


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RepliVAX is a novel single-cycle flavivirus (SCFV) vaccine platform, which contains a deletion in the capsid (C) gene of West Nile virus (WNV) that prevents the formation of infectious particles, unless the C gene is trans-complemented by a WNV C-expressing cell line. RepliVAX immunization results in a single round of infection, where the replication of genome leads to the production of subviral particles (SVP) and NS1 antigens that are highly immunogenic and induce an antiviral immune response without causing disease. The overall goal of this dissertation was to understand the interaction of RepliVAX with target cells, innate immune cells, and adaptive immune cells. The first studies investigated the properties of adaptive mutations acquired during the development of RepliVAX D2, which is a chimeric SCFV vaccine containing the dengue virus 2 (DENV2) prM/E genes in place of the corresponding WNV genes. It was demonstrated that mutations in the DENV prM/E region increased the specific infectivity of the chimeric virus particles. Also, it was shown that mutations in the WNV NS2A/NS3 region independently improved the encapsidation of virions without altering genome replication, indicating a functional interaction of structural and nonstructural flavivirus proteins to optimize the particle assembly/release of SCFV particles.
As RepliVAX mimics the infection of the wild type virus, inducing innate and adaptive immune responses, RepliVAX WN was utilized as a tool to further investigate different aspects of WNV immunity. The role of cellular innate immunity examined by macrophage depletion studies demonstrated that macrophages limit the initial dissemination of SCFV particles from the site of inoculation. Additionally, macrophages were not essential for activation of adaptive immune response, since equivalent CD8+ T cell response were found in depleted and non-depleted mice. This function was probably provided by dendritic cells (DC) and two subsets of DC were identified that stimulated WNV-specific T cell response. Finally, the role of type I interferon (IFN) was evaluated during SCFV immunization. It was shown that type I IFN response controls the early viral gene expression and consequently viral antigen production. However, the type I IFN did not affect the magnitude of the adaptive immune response, but did modulate effector cytokine production.



Flavivirus, WNV, RepliVAX, viral immunology, vaccine development