Abstract 2343 RNA phage Qβ display system for mapping SARS-CoV-2 spike protein functional domain and monitoring anti-S antibodies.

The motifs involved in tropism and immunological interactions of SARS-CoV-2 spike (S) protein were investigated utilizing the phage Qβ platform. We showed that separately, 7 overlapping peptide fragments representing the S protein (F1-7 of 200 residues each) could be inserted into the C-terminus of A1 on recombinant phage Qβ without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F3 from S425-525, was found to contain the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S442-510. Upstream, another fragment, F7, containing an overlapping portion of F4 would not bind to rhACE2, confirming not just only that residues were linear but equally also the appropriate structural orientation of F3 upon the phage Qβ. The F3/F4 (S450-608) and other inserts, including F4 (S611-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes, improved substantially the recognition and binding to their cognate antibodies. Additionally, we introduce a new biotin-binding peptide for detection and sensing of biotinylated proteins and nucleic acids. From a 15-mer library presented on the surface of the RNA phage Qβ, a HGHGWQIPVWPWGQG, 15 amino acids peptide longed nanotag could specifically bind to biotin. The combination of the chimeric epitope and the nanotag (15-AA), were monitored anti-S antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of point-of-care toolkit, subunit vaccines, or other biologics to combat SARS disease caused by SARS-CoV-2. This research was supported by the following agencies and grant funds: Grant number and Granting Agency (a) 1SC3GM132027-01A1; National Institutes of Health, National Institute of General Medical Sciences (NIH, NIGMS) - (b) BRG 2284736 and BRG2284729; CTSI /BRG - 2206945 BIOSENS-Biosensing; National Science Foundation (NSF) – (c) 1 R01 AG075132-01A1; NIH.