Combination attenuation offers strategy for live-attenuated coronavirus vaccines

Vineet D. Menachery; Lisa E. Gralinski; Hugh D. Mitchell; Kenneth H. Dinnon; Sarah R. Leist; Boyd L. Yount; Eileen T. McAnarney; Rachel L. Graham; Katrina M. Waters; Ralph S. Baric

Having addressed both heterologous challenge and efficacy in aged models, we next examined 235 the dNSP16/ExoN mutation for reversion potential. Similar to the original dNSP16 mutant, 236 dNSP16/ExoN showed no significant disease in RAG -/mice during acute infection. After 30 237 days, mice were euthanized, lung tissue harvested, homogenized, clarified, and used to infect 238 10-week old BALB/c mice. Contrasting dNSP16 (Fig. 4) The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/309591 doi: bioRxiv preprint weight loss or lethality upon acute infection (Fig. 7G) . Examination of input viral titer revealed 241 the absence of detectable virus in all five RAG -/mice infected with the dNSP16/ExoN mutant 242 (Fig. 7H) . The results indicate that following in vivo replication for 30 days, the dNSP16/ExoN 243 mutant failed to revert to a virulent form. In addition, the results suggest that the double mutant 244 could be cleared in immune compromised mice without the need for traditional adaptive 245 immunity. Together, the data argue that the dNSP16/ExoN mutant is unlikely to revert even in The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/309591 doi: bioRxiv preprint