Mapping Immunological, Host Receptor Binding Determinants, and Cathepsin Cleavage Site of EBOV Glycoprotein Utilizing the Qubevirus Platform

Ebola virus (EBOV) remains a highly infectious human pathogen that causes a severe and lethal disease known as Ebola virus disease (EVD), despite recent progress in vaccine development based on its only surface glycoprotein (GP). In this study, we modeled and inserted four overlapping fragments (F1-4) of the EBOV GP at the C-terminus of the A₁ protein of Qubevirus (Qβ) and used the platform to investigate the tropism and immunological functions of the GP by displaying the peptides with 30 overlapping amino acids. The resulting recombinant phages were used to determine their reactivity with GP-specific antibodies and their binding to the recombinant Niemann-Pick C1 (rNPC1) receptor in an immunoassay. In addition, modified, truncated, and C-terminus-tagged fragment F1 named F5 was utilized to map the cathepsin cleavage sites in an enzymatic assay. We demonstrated that a large GP peptide of 200 AA could be fused to A₁ and exposed on the Qβ platform in an accessible manner without significantly affecting its viability and infectivity. Fragments F1 (GP1-200), F2 (GP170-370), and F3 (GP350-550) were shown to contain important immune epitopes through binding to anti-GP-specific antibodies. Further, F1 was found to bind rNPC1, thereby suggesting a receptor binding determinant of the GP that was further confirmed in a competitive assay where the recombinant phages bearing the F1 fragment reduced the infectivity of EBOV pseudovirus by 27%. In addition, the viral infectivity was shown to be reduced by 46.39% by a cyclic peptide selected from an RNA Qβ library. Finally, F5 showed the cleavage sites to be AA191-192 and AA194-195 for CatB and L, respectively, which were further validated using a recombinant EBOV glycoprotein. These results provide insights into the antigenicity and tropism characteristic of the glycoprotein, with implications for the development of subunit vaccines or other biologics against Ebola virus disease.