Enhanced stability of the SARS CoV-2 spike glycoprotein trimer following modification of an alanine cavity in the protein core

Abstract

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity formed by Ala1016 and Ala1020 that form part of the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. The trimer stabilizing effects of filling the Ala1016/Ala1020 cavity was linked to improved S glycoprotein membrane fusion function. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited very high titers of neutralizing antibodies to ancestral and Delta-derived viruses (1/2,700-1/5,110), while neutralization titer was somewhat reduced with Omicron BA.1 (1/210-1,1744). The antigens elicited antibody specificities that could compete with ACE2-Fc for binding to the receptor-binding motif (RBM) and NAbs directed to key neutralization epitopes within the receptor-binding domain (RBD), N-terminal domain (NTD) and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S ectodomain trimers in the absence of an external trimerization motif (T4 foldon). The VI mutation represents a method for producing an intrinsically stable trimeric S ectodomain glycoprotein vaccine in the absence of a foreign trimerization tag.