Recombinant Engineering of SARS-CoV-2 Spike and N proteins

NIH Pandemic-Era Grants

Pandemic Era Grants

2023

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Principal Investigator: David  Margulies
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2023
Award: $66,875
Funding agency: National Institute of Allergy and Infectious Diseases

We have successfully engineered and produced a number of SARS-CoV-2 proteins, subunits, and single domain antibodies directed against the RBD. These purified proteins have provided the materials for obtaining high resolution crystal structures of four of these sybodies in complex with the Spike receptor binding domain (RBD) and one unliganded. In addition, we have addressed structural aspects of the sybody interactions with the complete Spike protein, using cryo-electron microscopy. We have extended our binding studies to evaluate the effects of SARS-CoV-2 variants of concern on the recognition by these sybodies. We have performed in silico mutagenesis of the RBD and molecular modeling to explain the results of our binding studies. We have applied our expertise in analyzing binding of antibodies to protein antigens to several additional anti-RBD antibodies produced in the Farci lab.  We have continued our study of antibodies to SARS-CoV-2 in two ways: 1) generating a set of bivalent, biparatopic antibody/peptide reagents that are being tested for inhibitory effects on viral adsorption; 2) developing computational tools and analysis of the accumulating large database of antibody/SARS complexes. Our computational analysis has recognized that there are some 23 sites on the surface of the SARS-CoV-2 RBD that may be recognized by different antibodies. These sites are characterized by a variety of protein secondary structural elements (helix, sheet, turn) and permit analysis of new viral variants with respect to how they may or may not be recognized by common antibodies.

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