Epistasis and background dependence in the evolution of Omicron variants of the SARS-CoV-2 spike protein

The rapid and repeated emergence of SARS-CoV-2 variants, particularly within the Omicron lineage, highlights the virus's remarkable ability to adapt under shifting immune pressures. A central molecular battleground in this evolutionary arms race is the spike receptor-binding domain (RBD), which must simultaneously maintain high affinity for the human ACE2 receptor while evading recognition by neutralizing antibodies. In this study, we construct and analyze multiple combinatorial libraries of SARS-CoV-2 RBD variants spanning major branches of Omicron evolution, including BA.1, BA.2, BA.5, XBB, and JN.1. Using high-throughput yeast display and binding assays, we map the effects of thousands of mutations and their combinations on ACE2 binding and antibody evasion. Our results reveal that while many RBD mutations exhibit additive effects, several mutations interact epistatically in a background-dependent manner. In particular, we identify synergistic interactions between BA.1 and BA.5 mutations that enhance antibody evasion, likely facilitating the rise of recombinant variants and convergent evolution. Conversely, some mutations show lineage-restricted compatibility, suggesting potential constraints on future evolutionary trajectories. Our comprehensive genotype-to-phenotype maps uncover both rugged and smooth regions of the viral fitness landscape and underscore the importance of epistasis in shaping SARS-CoV-2 evolution. These findings improve our ability to anticipate future viral variants and provide a framework for understanding how host-pathogen co-evolution unfolds at the molecular level.