Stabilizing Prefusion SARS-CoV-2 Spike by Destabilizing the Postfusion Conformation
Stabilizing Prefusion SARS-CoV-2 Spike by Destabilizing the Postfusion Conformation
Blog Article
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 hbl5266ca immunizations, the hamsters were challenged with live B.1.
351 SARS-CoV-2 virus for an evaluation of the protective efficacy.Results: 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 7gm pravana 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 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.