Our current work expands on previous papers we have published (Tan et al., 2010, PMID: 20087939; Barnwal et al., 2015, PMID: 25666762; Wu et al., 2016, PMID: 27633136), that characterize monoclonal antibodies (mAbs) interacting with non-structural protein 1 (NS1) of IAV as tools for research and diagnostic assay development. We further generated and characterized another novel mAb, named as 19H9, which binds to two highly conserved residues P85 and Y89 in NS1. Indeed, we demonstrate that mAb 19H9 exhibits broad cross-reactivity with IAV strains of different subtypes, in particular the newly-emerged highly pathogenic H5N1 and H5N6 avian strains. To our knowledge, mAb 19H9 is the first murine mAb to bind at the juxtaposition between the N-terminal RNA-binding domain and C-terminal effector domain of NS1. It could serve as a useful research tool for studying the conformational plasticity and dynamic changes in NS1.
Figure Legend: (A) Binding of mAb 19H9 to NS1(ED). NS1(RBD), NS1(ED) and full-length NS1 transfected cell lysates were subjected to IP with mAb 19H9 and protein A agarose beads followed by detection of immunoprecipitated proteins using rabbit anti-Myc antibody in Western blot. (B) Immunofluorescence showing subcellular localization of NS1 detected by mAb 19H9. (C) Epitope mapping of mAb 19H9. Western blot analysis of a series of N-, C-terminal truncation and full-length NS1 protein and (D) single substitution mutants of NS1(ED). (E) MAb 19H9 displays broad cross-reactivity with IAV strains of different subtypes. Western blot analysis of NS1 of H1N1, H3N2, H5N1, and H5N6 expressed in 293T cells was performed using either anti-Myc antibody (upper panel) or mAb 19H9 (lower panel).
Teo, Su Hui Catherine; Wu, Jian Ping; Mok, Chee Keng; Tan, Yee Joo. A NS1-Binding Monoclonal Antibody Interacts with Two Residues that are Highly Conserved in Seasonal as well as Newly-Emerged Influenza A Virus. Pathogens and Disease. Manuscript in review.