Development of a molecular system to identify variants of the SARS-CoV-2 S glycoprotein that enhance cellular attachment

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative intracellular pathogen of the coronavirus disease 2019 (COVID-19) pandemic. The unprecedented strain on health care systems worldwide continues to impact public health efforts to contain the viral spread. Although vaccines are designed against SARS-CoV-2, their efficiency against emerging SARS-CoV-2 variants of concern appears to be reduced. Variants of concern encode S glycoprotein amino acid changes which can increase viral fitness determined through immune evasion, transmissibility, and viral entry. This study trialled and tested a molecular system for the identification of S glycoprotein variants that can bind to angiotensin converting enzyme 2 (ACE2), the host cellular receptor, with higher binding affinity compared with the ancestral SARS-CoV-2 strain. S glycoprotein variants were generated by combinatorial mutagenesis libraries spanning two amino acid regions of the S glycoprotein receptor-binding domain. The combinatorial libraries showed high levels of degeneracy at the expected amino acid positions. Lentiviral vectors pseudo-typed with the SARS-CoV-2 S glycoprotein combinatorial mutagenesis libraries were produced and viral RNA was extracted for sequencing. HEK293T and hACE2-HEK293T cell lines were transduced with pseudo-typed lentivirus (pseudo-LV) into a transgene cell line to confirm the S glycoprotein interacted with host receptor ACE2. Blasticidin selection after 12 days confirmed the integration of the viral RNA into the hACE2-HEK293T cell genome, but not into the HEK293T cells demonstrating the successful production of S glycoprotein pseudo-LV. This study sought to capture S glycoprotein variants that bind ACE2-Fc protein tagged to Protein A Dynabeadsä with high affinity and eluting the captured S glycoproteins to identify any protein-coding changes which frequently arise and may preferentially bind ACE2. The results of this study hope to contribute to the development of a novel molecular system to screen S glycoprotein variants prior to their circulation in the community. Ultimately the method developed here, if applied at an industrial scale, could be used for the design of multivalent mRNA vaccines immunising populations to prevent the health consequences likely to be caused by variants yet to evolve.