Browsing by Author "Salunke, Dinakar M."

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Epitope-directed anti-SARS-CoV-2 scFv engineered against the key spike protein region could block membrane fusion
(John Wiley & Sons, 2023) Jaiswal, Deepika; Kumar, Ujjwal; Gaur, Vineet; Salunke, Dinakar M.
The newly emerged SARS-CoV-2 causing coronavirus disease (COVID-19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2' site, by the TMPRSS2 protease. S2' cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2' site to be conserved among all the SARS-CoV-2 variants, we chose an S2' epitope encompassing the S2' cleavage site and generated single-chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2' epitope was determined. Incidentally, S2' epitope in the scFv bound structure adopts an alpha-helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody-S2' epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2' cleavage competitively.
A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein
(BioMed Central Ltd, 2023) Kumar, Ujjwal; Goyal, Priya; Madni, Zaid K.; Kamble, Kajal; Gaur, Vineet; Rajala, Maitreyi S.; Salunke, Dinakar M.
Background: The influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation. Methods: The phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging. Results: The four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells. Conclusion: This structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.