High Affinity Human Antibody Fragments to Dengue Virus Non-Structural Protein 3

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 5′ 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 naïve 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 α3″ (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 valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

Author Summary

Dengue virus is the most prevalent mosquito transmitted infectious disease in humans and is responsible for febrile disease such as dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Dengue non-structural protein 3 (NS3) is an essential, multifunctional, viral enzyme with two distinct domains; a protease domain required for processing of the viral polyprotein, and a helicase domain required for replication of the viral genome. In this study ten unique human antibody fragments (Fab) that specifically bind dengue NS3 were isolated from a diverse library of Fab clones using phage display technology. The binding site of one of these antibodies, Fab 3F8, has been precisely mapped to the third α-helix within subdomain III of the helicase domain (amino acids 526–531). The antibody inhibits the helicase activity of NS3 in biochemical assays and reduces DENV replication in human embryonic kidney cells. The antibody is a valuable tool for studying dengue replication mechanisms.