Simulation-Driven Design of Stabilized SARS-CoV-2 Spike S2 Immunogens

The full-length prefusion-stabilized SARS-CoV-2 spike (S) is the principal antigen of COVID-19 vaccines. Vaccine efficacy has been impacted by emerging variants of concern that accumulate most of the sequence modifications in the immunodominant S1 subunit. S2, in contrast, is the most evolutionarily conserved region of the spike and can elicit broadly neutralizing and protective antibodies. Yet, S2’s usage as an alternative vaccine strategy is hampered by its general instability. Here, we use a simulation-driven approach to design S2-only immunogens stabilized in a closed prefusion conformation. Molecular simulations provide a mechanistic characterization of the S2 trimer’s opening, informing the design of tryptophan substitutions that impart kinetic and thermodynamic stabilization. Structural characterization via cryo-EM shows the molecular basis of S2 stabilization in the closed prefusion conformation. Informed by molecular simulations and corroborated by experiments, we report an engineered S2 immunogen that exhibits increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses. ### Competing Interest Statement J.S.M., L.Z., R.E.A., X.N., L.C., M.S. are inventors on a U.S. patent application describing the use of stabilized SARS-CoV-2 S proteins as vaccine antigens (Stabilized SARS-CoV-2 S Antigens, 63/583,090). K.C. is a member of the scientific advisory board of Integrum Scientific LLC and has consulted for Axon Advisors, LLC. K.C. owns shares in Integrum Scientific and Eitr Biologics, Inc. Trajectories of S2 trimer opening pathways, WE configuration files, and representative closed prefusion conformations of HexaPro-SS-2W are included in the article as Supplementary Data 1. Full datasets (simulation input files and trajectories) for conventional MD, GaMD, WE MD, and free energy calculations are available for download on the Amaro Lab website . Source data are provided with this paper. The atomic coordinates of HexaPro-SS-2W in the closed prefusion conformation are available in the Protein Data Bank under PDB ID: 8VAO. The cryo-EM map is available from the Electron Microscopy Data Bank under accession code EMDB-43097.