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

Stabilizing Prefusion SARS-CoV-2 Spike by Destabilizing the Postfusion Conformation
type: AcademicArticle
type: Article
abstract: 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: 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 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.
date: 2025
volume: 13
issue: 3
author list: authors
type: EPrint
type: ArticleEPrint
is part of: repository

Vaccines
type: Collection

Raju S. Rajmani
type: Person
given name: Raju S.
family name: Rajmani

Debajyoti Chakraborty
type: Person
given name: Debajyoti
family name: Chakraborty

Randhir Singh
type: Person
given name: Randhir
family name: Singh

Raghavan Varadarajan
type: Person
given name: Raghavan
family name: Varadarajan

Rajesh P. Ringe
type: Person
given name: Rajesh P.
family name: Ringe

Sahil Kumar
type: Person
given name: Sahil
family name: Kumar

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