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

Abstract

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, the usage of S2 as an alternative vaccine strategy is hampered by its general instability. Here, we use a simulation-driven approach to design highly stable S2-only antigens retaining a closed prefusion conformation. Weighted ensemble simulations provide mechanistic characterization of the S2 trimer opening, informing the design of tryptophan substitutions that impart kinetic and thermodynamic stabilization. Alchemical free energy perturbation calculations and a corroborating set of experiments confirm that V991W and T998W in the central helices of S2 stabilize the trimer in the closed prefusion conformation, producing an antigen with increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses.

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“…#UFC905096). SARS-CoV-2 (Wuhan-1 strain) S2 protein (Hexapro-SS-2W) included residues 697-1208 of the spike with an artificial signal peptide, proline substitutions at positions 817, 892, 899, 942, 986, and 987, cysteine substitutions at positions 704 and 790, a glutamate substitution at 957, tryptophan substitutions at 991 and 998, and a C-terminal foldon trimerization motif, HRV 3C cleavage site, 8× His tag and Twin-Strep-tag 51 . Soluble Hexapro-SS-2W protein was expressed and purified as described 51 in FreeStyle 293F cells (ThermoFisher, Cat.…”