West Nile virus (WNV) has a worldwide distribution, with this virus having been isolated on all continents except Antarctica. The recent emergence of highly pathogenic strains of WNV associated with increased rates of neurological disease is of great concern given this broad distribution of the virus. Although two vaccines have been licensed for veterinary use, no prophylactic measures have been approved for humans. Similarly, no antivirals are currently available for post-exposure treatment of WNV. Indeed, few therapeutic agents have shown promise when administered after WNV infection in animal models. KUNV is a highly attenuated, Australasian lineage 1 strain of WNV. This attenuation is mediated in part by the limited neuroinvasiveness of this virus. Phylogenetically, KUNV clusters with pathogenic lineage 1 WNV strains, including the isolates which have been associated with 997 deaths in North America since 1999. Recently, it was shown that mice exposed to KUNV were effectively protected from challenge with pathogenic WNV. The KUNV strain used in that study possessed a single amino acid substitution in NS1 protein that affected oligomerization of this protein, resulting in reduced virus replication in vitro and increased attenuation in mice. In the present study, further characterization of this attenuation marker in NS1 protein was undertaken to determine whether it is suitable for inclusion in a live-attenuated KUNV vaccine. Similarly, mapping of the residues that contribute to the dimerization domain surrounding NS1 protein was performed to identify other potential attenuation markers for stabilization of KUNV attenuation. The mutant viruses created in this study also were manipulated to characterize the role of NS1 protein dimerization in flavivirus replication. The results of this work indicate that NS1 protein dimerization is not absolutely required for virus replication or production of secreted oligomers of NS1 protein, which are important for eliciting protective humoral responses. Although replication of KUNV was found to be highly dependent on retention of the conserved amino acid sequence within the dimerization domain, two mutant viruses were generated by introducing substitutions at residue 250 of NS1 protein. The resultant viruses demonstrated reduced replication in vitro and attenuation in mice. Similarly, a non-conservative substitution in NS2A, which was previously shown to reduce the resistance of KUNV to the host interferon response, was able to attenuate KUNV in mice. Inoculation of adult mice with viruses containing mutations at either site afforded complete protection from lethal WNV challenge. However, the substitutions described in the dimerization domain of NS1 protein were unstable, with restoration of virulence being observed in mutant viruses after limited passaging in vitro. Concerns over the stability of attenuating mutations in KUNV and the time taken to characterize new attenuation markers prompted the evaluation of a novel approach to the development of rationally-designed flavivirus vaccines. The introduction of large complements of synonymous codon substitutions reduced KUNV replication in vertebrate cells. Escape mutations were not observed in a KUNV vaccine candidate containing 37 rare codons after repeated passaging in vertebrate cells at a low MOI. Replication of KUNV in C6/36 cells was unaffected by the introduction of large numbers of rare codons, indicating that this cell line exerts limited selective pressure on the codon composition of this virus. This observation indicates that C6/36 cells may be a useful cell line for the propagation of viruses containing this type of mutation. Finally, three monoclonal antibodies (MAbs) which bind to WNV envelope (E) protein were observed to potently neutralize the pathogenic NY99 strain of WNV. Passive administration of one of these antibodies was shown to afford mice protection even when administered seven days after challenge with WNV NY99 strain. Remarkably, this is the same time that mortality is first observed in control groups. These antibodies mapped to the putative receptor binding domain (domain 3) of E protein. However, these antibodies were found to block virus replication at a stage after receptor-binding. Homology modeling was used to propose a mechanism for the blockade of virus infection mediated by MAb binding. This study describes the development and characterization of a promising new vaccine as well as candidate immunotherapeutics for the prophylaxis and post-exposure treatment of WNV disease. This work described herein also has implications for the development of vaccines and antivirals for other flaviviral diseases.
| Identifer | oai:union.ndltd.org:ADTP/253936 |
| Creators | Mr David Clark |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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