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

Unlike the ExoN and E protein mutants, dNSP16 coronaviruses demonstrate no significant 101 attenuation in terms of viral replication in vitro except in the context of type I IFN pretreatment 102 (19) . Similarly, in vivo characterizations found no deficit in viral replication at early times (days 1 103 and 2), but a marked attenuation at late times post infection (19, 20) . These results suggest that 104 the early portions of dNSP16 infection are equivalent to wild-type virus infection. To further 105 explore this question, we utilized a systems biology approach to compare wild-type and dNSP16 106 mutant in 20-week old C57BL/6 mice. In this model, mice infected with the mouse-adapted mutant has no discernable attenuation at days 1 or 2 (Fig. 1C) ; however, at day 4, a 100-fold 111 reduction in replication is observed in the lungs of dNSP16 infected mice that corresponds with 112 the kinetics of interferon stimulated gene (ISG) expression. Importantly, the global host 113 expression response models the same trend. PCA plots of total RNA expression finds that wild-114 type and mutant virus profiles cluster together at days 1 and 2 ( Fig. 1D) dNSP16, monitored for 28 days, and subsequently challenged with the chimeric strain following vaccination (Fig. 2D) . Together, the data indicate that a live-attenuated NSP16 137 vaccine has sufficient capacity to protect from heterologous CoV challenge in young mice.