title: Identification and molecular characterization of human antibody fragments specific for dengue NS5 protein.
abstract: The multifunctional dengue nonstructural (NS) protein 5 from the four serotypes of dengue virus (DENV1-4) is essential for viral replication and harbors a methyl transferase (MTase) and a RNA-dependent RNA-polymerase domain (RdRp). There are limited comparative studies of NS5 from the four DENV serotypes and this is further hampered by a lack of cross-reactive NS5 antibodies. In this study, recombinant NS5 proteins were expressed, purified, enzymatically characterized, and used strategically as bait in biopanning experiments with a naïve human Fab phage-display library to identify serotype specific or cross-reactive Fab fragments. Using a combination of peptide competition ELISA and peptide phage display the epitopes of the cross-reactive Fabs were mapped to the first alpha helix of the MTase domain (5M1) and the priming loop of the RdRp domain (5R3). The epitope of a third, serotype-specific Fab (5M3) was mapped to aa19-30 of the DENV3 MTase domain. Together the recombinant proteins and specific antibodies will facilitate further mechanistic studies of the DENV replication complex.
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore.
authors.forename: Yongqian
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore.
authors.forename: Nicole J
authors.initials: NJ
authors.lastname: Moreland
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore.
authors.forename: Moon Y F
authors.initials: MY
authors.lastname: Tay
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore.
authors.forename: Chin Chin
authors.initials: CC
authors.lastname: Lee
authors.affiliation: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore; Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
authors.affiliation: Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore. Electronic address: subhash.vasudevan@duke-nus.edu.sg.
authors.forename: Subhash G
authors.initials: SG
authors.lastname: Vasudevan
mesh_terms: D000595:Amino Acid Sequence; D003429:Cross Reactions; D003715:Dengue / Q000276:immunology / Q000821:virology*; D003716:Dengue Virus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D018604:Epitope Mapping; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000235:genetics / Q000276:immunology; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D013045:Species Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014779:Virus Replication
chemical_list: D007140:Immunoglobulin Fab Fragments; C582223:NS5 protein, dengue virus; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; D012324:RNA-Dependent RNA Polymerase
keywords: Antibody; Cross-reactivity; DENV; Dengue; Fab; MTase; NS; NS5; NS5FL; Phage display; RNA-dependent RNA polymerase; RdRp; antigen-binding fragment; dengue virus; full-length NS5 protein; methyltrasferase; non-structural protein