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Career Development and Training Program

Individual Project CD008

Collaborating Institution: University of Texas Medical Branch (UTMB), Galveston, TX

Principal Investigator: Naomi Forrester, PhD

Title of the Project: Using High-fidelity Mutations to Stabilize Live-attenuated Vaccines

Mentors:

Expected Product: Arbovirus vaccine constructs with increased replication fidelity to prevent mutations without compromising vaccine effectiveness.

Description: Effective, stable vaccines are necessary for combating arboviral diseases. The most effective vaccines are live-attenuated constructs that are cheap to produce and able to deliver effective, long-lasting immunity with a single dose, of importance in resource-poor countries. One major obstacle to the production of stable live-attenuated vaccines for RNA viruses is the inherent plasticity in RNA viral genomes. RNA viruses lack a proof-reading domain which results in increased mis-incorporation of mutations. This inherent plasticity of the genomes means that there is always a risk of mutations, either reversion or compensatory, that generates the wild-type phenotype or enhanced virulence, with the potential to instigate the very epidemic the vaccine was supposed to prevent in the first place. Recently, there has been increased interest in this aspect of vaccine development, in part due to the deep sequencing technologies that allow sequencing of multiple RNA strands rather than just the consensus sequence. This is particularly true for arboviral diseases where the vaccine has to show stability and attenuation in both the vertebrate and mosquito host. Recently, mutations have been identified in the RNA-dependent RNA-polymerase (RdRp) that increase fidelity in poliovirus (PV), chikungunya virus (CHIKV) and West Nile virus (WNV). Engineering these mutations into vaccine constructs will increase the vaccine stability as the increased fidelity prevents mis-incorporations that result in reversion of the engineered mutations and prevents incorporation of new mutations that would result in increased virulence. The goal of this proof of concept study is to characterise mutations identified in the CHIKV and Venezuelan equine encephalitis virus (VEEV) RdRp that have been preliminarily determined to enhance fidelity, reducing the viral diversity by ~30% as well as reducing the pathogenicity of the constructs ~ 50%. These mutations will be engineered into well-characterized vaccine constructs, 181/clone 25 for CHIKV and TC-83 for VEEV and evaluated for stability, immunogenicity and virulence in appropriate mouse models. In addition the flavivirus yellow fever vaccine (YF VaxTM) will be passaged in the presence of nucleotide analogues to identify possible mutations associated with high-fidelity replication. Identifying mutations that increase fidelity will lead to more stable live-attenuated vaccines. For the arboviruses, which are increasing in incidence, increasing the stability, without compromising the effectiveness of the vaccine will be highly beneficial.