Characterization of a neutralizing monoclonal antibody binding to a conserved epitope in the vestigial esterase domain of hemagglutinin of H5Nx influenza A viruses
Yee-Joo Tan1,2, Subha Sankar Paul1, Nur Khairiah Binte Mohd Ismail1, Zheng Zhiqiang1 1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore; 2Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
Since the first human infection of H5N1 influenza A virus in 1997, the virus has evolved and reassorted to give rise to different clades of H5N1 as well as H5Nx viruses. In May 2014, the first human case of avian H5N6 IAV infection was identified in China's Sichuan Province (http://www.wpro.who.int/china/mediacentre/releases/2014/20140507/en/). Subsequently, human infections were confirmed in different parts of China. Previously, a murine monoclonal antibody (MAb) 9F4 was generated against clade 1 H5N1 hemagglutinin (HA) and shown to have cross-clade neutralizing efficacy and inhibit viral entry by preventing the pH-mediated conformational change of HA. In this study, through escape mutant generation and in-silico prediction, it was revealed that MAb 9F4 binds to a novel epitope in the vestigial esterase (VE) sub-domain of HA comprising at least three non-continuous amino acid residues, arginine (R) at position 62, tryptophan (W) at position 69 and phenylalanine (F) at position 79, which interacted with MAb 9F4 in a conformation-dependent manner. Sequence alignment revealed that two residues in the MAb9F4 binding epitope are conserved in two strains of H5N6 (A/Guangzhou/39715/2014 (H5N6) and A/Sichuan/26221/2014 (H5N6)) isolated from patients. However, the arginine R62 has been substituted with K which is a basic amino acid like R. Consistently, our results showed that MAb 9F4 can prevent the entry of H5N6 pseudotyped virus into host cells. Competitive binding also revealed that the binding epitope of MAb 9F4 is distinct from 3 other neutralizing MAbs known to bind to VE. Thus far, only 1 VE-binding MAb was shown to require Fc-FcγR interaction for in vivo protection. Hence, we are currently investigating if protection conferred by MAb 9F4 in a mouse model of infection is dependent on Fc-FcγR interaction.
Funding: This work was supported by a grant from the Ministry of Education (MOE) of Singapore [AcRF Tier 2, grant no. MOE2015-T2-2-052].
Credits: None available.
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