high affinity human antibody fragments to dengue virus non-structural protein 3人类高亲和力抗体片断登革病毒非结构蛋白3.pdfVIP

High Affinity Human Antibody Fragments to Dengue Virus Non-Structural Protein 3 1. 1. 1 1 1 Nicole J. Moreland , Moon Y. F. Tay , Elfin Lim , Prasad N. Paradkar , Danny N. P. Doan , Yin Hoe 2 2 1 Yau , Susana Geifman Shochat , Subhash G. Vasudevan * 1 Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, Singapore, Singapore, 2 School of Biological Sciences, Nanyang Technical University, Singapore, Singapore Abstract Background: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 59 triphosphatase domain which forms the remainder of the 618-aa long protein. Methodology/Principal Findings: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently ¨ attached via a flexible linker, was used as bait in biopanning with a naıve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. Conclusions/Significance: Antibodies such as 3F8 are