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

• Published in: PLoS pathogens Vol. 19; no. 5; p. e1010981
• Main Authors: Poumbourios, Pantelis, Langer, Christine, Boo, Irene, Zakir, Tasnim, Center, Rob J, Akerman, Anouschka, Milogiannakis, Vanessa, Aggarwal, Anupriya, Johnstone, Bronte A, Ha, Jungmin, Coulibaly, Fasséli, Turville, Stuart G, Drummer, Heidi E
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.05.2023; Public Library of Science (PLoS)
• Subjects: Alanine; Amino acids; Angiotensin-Converting Enzyme 2; Antibodies; Antibodies, Neutralizing; Antigenicity; Antigens; Biology and Life Sciences; Cell fusion; Cell membranes; Chromatography; Coils; Coronaviruses; COVID-19; COVID-19 vaccines; Disease transmission; Domains; Glycoproteins; Health aspects; HIV; Human immunodeficiency virus; Humans; Hydrophobicity; Infections; Medicine and Health Sciences; Membrane fusion; Mutants; Mutation; Neutralizing; Oligomers; Pandemics; Peptides; Physiological aspects; Proteins; Research and Analysis Methods; Residues; Severe Acute Respiratory Syndrome; Severe acute respiratory syndrome coronavirus 2; Spike glycoprotein; Spike Glycoprotein, Coronavirus; Structure; Thermal stability; Trimers; Vaccine efficacy; Vaccines; Viral antibodies; Viral diseases; Viruses; Denmark; Australia
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1010981

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 subunit 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 next to Ala1016 and Ala1020 in 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. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.