Stabilizing Prefusion SARS-CoV-2 Spike by Destabilizing the Postfusion Conformation

Stabilizing Prefusion SARS-CoV-2 Spike by Destabilizing the Postfusion Conformation

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Background/Objectives: As with many viral fusion proteins, the native conformation of SARS-CoV-2 Spike is metastable.Most COVID-19 vaccines utilize a stabilized Spike (Spike-2P) containing two proline substitutions, and subsequently, a further stabilized variant with four additional proline substitutions, Spike-6P, has been developed.In an alternative approach, we introduced two aspartic acid residues (2D) in the HR1 region of Spike at positions that are exposed and buried in the pre- and postfusion states, respectively, to destabilize the postfusion conformation.Methods: The recombinant protein constructs were expressed in a mammalian cell culture and characterized for their yield and antigenicity, and the formulations were then used to immunize hamsters.After two immunizations, the hamsters were challenged with live B.

1.351 SARS-CoV-2 virus for an evaluation of the protective efficacy.Results: Left Crank The introduction of the two aspartic acid mutations resulted in an approximately six-fold increase in expression, comparable to that in Spike-2P.When the 2D mutations were combined with the above four proline mutations (Spike-4P-2D), this led to a further three- to four-fold enhancement of protein expression, similar to that seen in Spike-6P.When formulated with the oil-in-water emulsion adjuvant Sepivac SWE, the 2P, 2D, 6P, and 4P-2D Spike variants all protected female hamsters against heterologous challenge with the B.

1.351 SARS-CoV-2 virus and elicited high titers of neutralizing antibodies.Conclusions: We suggest that destabilization of the postfusion conformation through the introduction Studs of charged amino acids at sites that are exposed in the pre- and buried in the postfusion conformation offers a general strategy to enhance the yield and stability of the native, prefusion conformation of viral surface proteins.