Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM Wednesday Speaker Abstracts

HARNESSING MOLECULAR SIMULATIONS TO DESIGN STABILIZED SARS-COV 2 S2 ANTIGENS Lorenzo Casalino 1 ; Xandra Nuqui 2 ; Ling Zhou 3 ; Mohamed Shehata 1 ; Albert Wang 6 ; Alexandra L Tse 6 ; Anupam A Ojha 2 ; Fiona L Kearns 1 ; Mia A Rosenfeld 1,4 ; Emily Happy Miller 6,7 ; Cory M Acreman 3 ; Surl-Hee Ahn 5 ; Kartik Chandran 6 ; Jason S McLellan 3 ; Rommie E Amaro 1,2 ; 1 University of California, San Diego, Molecular Biology, La Jolla, CA, USA 2 University of California, San Diego, Chemistry and Biochemistry, La Jolla, CA, USA 3 The University of Texas at Austin, Molecular Biosciences, Austin, TX, USA 4 National Institutes of Health, Laboratory of Computational Biology, National Heart, Lung and Blood Institute, Bethesda, MD, USA 5 University of California Davis, Chemical Engineering, Davis, CA, USA 6 Albert Einstein College of Medicine, Microbiology and Immunology, Bronx, NY, USA 7 Albert Einstein College of Medicine, Department of Medicine, Division of Infectious Diseases, Bronx, NY, USA The effectiveness of COVID-19 vaccines, rooted in the full-length prefusion-stabilized SARS CoV-2 spike protein, is challenged by the continual emergence of variants of concern accumulating sequence modifications in the immunodominant S1 subunit. A possible solution to this limitation lies in the spike's S2 subunit, known for its evolutionary conservation across sarbecoviruses and ability to elicit a robust immune response. Yet, the inherent instability of S2 remains a significant hurdle to its application in vaccine design. To tackle this challenge, we used weighted ensemble molecular dynamics simulations to examine the conformational plasticity of the S2 trimer and inform the design of tryptophan cavity-filling mutations aimed at stabilizing the trimer in a closed prefusion conformation. Alchemical non-equilibrium free energy calculations of three engineered S2 variants in the closed state revealed a stabilizing energetic contribution imparted by the tryptophan substitutions. Experimental assays, including cellular expression and differential scanning fluorimetry, validated the computational predictions by demonstrating increased expression yields and enhanced thermostability for the engineered S2 variants. Furthermore, these stabilizing mutations facilitated the determination of a high resolution cryo-EM structure of the S2 trimer in its closed prefusion conformation, revealing an extended network of new, stabilizing hydrophobic interactions also observed in the simulations. Finally, we characterized the immunogenicity of the engineered S2 antigen, demonstrating its ability to elicit neutralizing responses against sarbecoviruses. Leveraging a simulation-driven approach and supported by experimental and cryo-EM structural data, our findings pinpoint specific cavity-filling substitutions that enhance the stability of the SARS-CoV-2 S2 trimer, making it a promising antigen that could potentially be incorporated into viable vaccine platforms.

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