Abstract
SARS-CoV-2 Omicron rapidly outcompeted other variants and currently dominates the COVID-19 pandemic. Its enhanced transmission and immune evasion are thought to be driven by numerous mutations in the Omicron Spike protein. Here, we systematically introduced BA.1 and/or BA.2 Omicron Spike mutations into the ancestral Spike protein and examined the impacts on Spike function, processing, and susceptibility to neutralization. Individual mutations of S371F/L, S375F, and T376A in the ACE2-receptor-binding domain as well as Q954H and N969K in the hinge region 1 impaired infectivity, while changes to G339D, D614G, N764K, and L981F moderately enhanced it. Most mutations in the N-terminal region and receptor-binding domain reduced the sensitivity of the Spike protein to neutralization by sera from individuals vaccinated with the BNT162b2 vaccine and by therapeutic antibodies. Our results represent a systematic functional analysis of Omicron Spike adaptations that have allowed this SARS-CoV-2 variant to dominate the current pandemic.