Pressures to combat the coronavirus disease 2019 (COVID-19) pandemic have led to accelerated efforts to develop vaccines and therapeutics. The spike (S) protein, which is a key target of these efforts is metastable and challenging to recombinantly generate. Using a structure-guided approach, we characterized 100 spike designs, 26 of which improved protein yields. Combinations of the beneficial substitutions led to the development of HexaPro, a S protein variant with a 10-fold higher expression than the parental construct enhanced thermal-stability, due to six proline substitutions. Further structural and antigenic characterization of HexaPro confirmed a prefusion stabilized S protein with conserved ACE2 and antibody binding. The improved yield and stability of HexaPro has accelerated the development of serological diagnostics and vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Spike is also undergoing immunogenic selection with variants that increase infectivity and partially escape convalescent plasma. I developed Spike Display, a high-throughput platform to rapidly characterize glycosylated S ectodomains across multiple coronavirus-family proteins. I assayed ∼200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in the alpha, beta, gamma, epsilon, and delta impact spike expression and escape most NTD-targeting nAbs. Finally, beta and gamma completely escaped a potent ACE2 mimic. The continuous spread and evolution of SARS-CoV-2 has led to the repeated emergence of variants of concern. For the Omicron variant, sub-lineages BA.1 and BA.2 respectively contain 33 and 29 nonsynonymous and indel spike protein mutations. These amino acid substitutions and indels are implicated in increased transmissibility and enhanced immune evasion. By reverting individual Spike mutations of BA.1 or BA.2, I characterized the molecular effects of the Omicron spike mutations on expression, ACE2 receptor affinity, and neutralizing antibody recognition. I identified key mutations enabling escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein can compensate for destabilizing mutations in the receptor binding domain, enabling the record number of mutations in Omicron. My results provide a comprehensive account of the mutational effects in the Omicron spike protein and illuminate previously uncharacterized mechanisms of host evasion. I anticipate that Spike Display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.Cellular and Molecular Biolog
