A yellow fever 17D and Usutu virus chimera with rationally designed mutations in the envelope protein is lethal in an...

The expanding geographical spread of Usutu virus (USUV) poses an increasing threat to bird populations and to human health. Here we assessed a chimeric USUV vaccine candidate that was constructed using the live attenuated yellow fever virus (YFV) YF-17D platform. In our chimeric virus, the pre-membrane (PrM) and envelope (E) proteins of YFV were replaced with those of USUV. Similar chimeras of YF-17D with otherflaviviruses have been reported to be attenuated in vivo, making them promising modified live virus vaccine candidates. We used either wild type USUV PrME or USUV PrME containing rationally designed mutations in the E protein that were expected to (further) attenuate the virus, based on their effect in the context of other orthoflaviviruses. In cell culture the YF-17D/USUV chimeric viruses displayed reduced fitness compared to both YF-17D and USUV, with the chimera containing the mutated USUV E exhibiting the slowest growth kinetics. However, in our interferon α/β receptor deficient (Ifnar^-/-) mouse model the chimeric YF-17D/USUV viruses caused rapid lethality in all animals, with survival times that were only marginally better compared to wild type USUV. It was surprising that introduction of the PrME region from the naturally low pathogenic USUV into the YF-17D platform yielded viruses that caused lethal infections in our model, while in contrast other neuroinvasive orthoflaviviruses are attenuated in this same chimeric platform. All control mice that were infected with a 1000-fold higher dose of YF-17D survived. We also discovered that the mutations in E that are attenuating in related viruses did not result in attenuation in the Ifnar^-/- mouse model, neither in the context of USUV itself or the YF-17D chimera. This may be due to the immunocompromised nature of the mice or because these mutations are not attenuating in the context of USUV. This work highlights some complications and unpredictability related to the use of the chimeric live attenuated YF-17D vaccine platform and introduction of rationally designed mutations with a predicted attenuating effect. Furthermore, our work illustrates that infection models that are able to better predict the phenotype and safety of (chimeric) USUV/orthoflavivirus vaccine candidates are needed.