Non-coding flavivirus RNA as key target to engineer live-attenuated vaccines that are ‘safe-by-design’

Viruses in the Flavivirus genus, such as dengue, Zika, West Nile (WNV) and tick-borne encephalitis (TBEV), can cause severe illness in humans. Due to their extensive spread in recent years, they pose a significant public health threat. In particular, WNV and TBEV are on the rise in Europe. For few flaviviruses effective vaccines are available. We have identified a key target that enables the ‘safe-by-design’ engineering of novel flavivirus vaccines. Our research and that of others has demonstrated that all flaviviruses produce abundant quantities of small non-coding RNA required for induction of cytopathic effect in cells and for viral pathogenicity in vivo. This so-called subgenomic flavivirus RNA (sfRNA), is the product of incomplete viral genomic RNA degradation by the cellular exoribonuclease XRN1 that stalls at conserved 3D RNA structures in the 3’ untranslated region (UTR). Despite the increasing global risk of flavivirus infections, the mechanism of sfRNA-mediated pathogenicity is poorly understood and for many flaviviruses no safe and effective vaccines have been developed. Therefore, this research aims to elucidate the mechanism of sfRNA-induced pathogenicity and engineer live-attenuated flavivirus vaccines based on the elimination of sfRNA production. In-depth knowledge on the molecular mechanism of virus attenuation is essential to successfully license novel vaccine candidates via regulatory authorities. In this project, WNV and TBEV vaccine candidates will be generated by construction of sfRNA-deficient mutant viruses. This will be done by mutagenesis of defined three-dimensional RNA structures within the 3’UTR that are essential for sfRNA biogenesis. An innovative deep mutational scanning approach will be applied to generate a library of mutants, which will be phenotypically selected in a high-throughput manner. Selection is based on their ability to efficiently replicate yet fail to induce CPE in cell culture, while their immunogenicity and safety will be studied in mice. It is hypothesized that single-shot vaccination of mice with the sfRNA-deficient viruses will induce a highly protective, long lasting immune response, because the viruses can still replicate but no longer cause disease. The mechanism by which sfRNA determines pathogenicity will be elucidated by analysis of its effect on apoptosis (programmed cell death). Involvement of sfRNA in two key apoptotic pathways will be analysed to untangle the exact mechanism by which sfRNA normally targets cellular components resulting in viral pathogenicity. Ultimately, a fundamental understanding of flavivirus pathogenicity and the licensure of safe and effective flavivirus vaccines is important for management of current and emerging flavivirus outbreaks.