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  current news   Press   selected story    
     
  20 July 2017  
 
A cross-clade H5N1 influenza A virus neutralizing monoclonal antibody binds to a novel epitope within the vestigial esterase domain of hemagglutinin
 
 




Authors
Subha Sankar Paula,1, Chee-Keng Moka,1, Tze-Minn Mak b, Oi-Wing Ng a,
James Odame Aboagyea,c, Teddy John Wohlboldd,e, Florian Krammerd, Yee-Joo Tana,c,*.

a  Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University    Health System (NUHS), National University of Singapore, Singapore
b  NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore,    Singapore
c  Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research),    Singapore
d  Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, USA
e  Graduate School of Biological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA

Published online in Antiviral Research on 19 June 2017.

Abstract
The sporadic outbreaks of highly pathogenic H5N1 avian influenza virus have raised public health concerns. Monoclonal antibodies (MAbs) against hemagglutinin (HA) have been increasingly used successfully for therapeutic purposes. Previously, MAb 9F4, generated against clade 1 H5N1 HA, was observed to have cross-clade neutralizing efficacy and inhibited viral entry by preventing the pHmediated conformational change of HA. Furthermore, mouse-human chimeric MAb 9F4 was found to retain high degrees of neutralizing activity. 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 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. Binding and neutralization studies suggested that R62 is the critical residue for MAb 9F4 binding whereas W69 and F79 seem to cooperate with R62 to stabilize the epitope.
Mutation of either R62 or W69 did not affect replicative fitness of the virus in vitro. Interestingly, MAb
9F4 retained neutralizing efficacy against a clade 2.3.2.1a H5N1 virus consisting of an arginine to lysine
substitution at position 62 in HA.

Figure

Figure legend

Quantitative comparison of the interaction of MAb 9F4 with VN04-HA mutants.
(A) An immunofluorescence assay, as described before, was carried out on H1299 cells expressing empty vector, VN04-HA-WT, or VN04-HA-W69A. Staining with MAb 9F4 and anti-HA5-C PAbwas performed on the same permeabilized H1299 cells. Overlay of MAb 9F4 and anti-HA5-C PAb binding is shown along with nuclei counterstained with DAPI. (B), (C) H1299 cells were transfected with empty vector or plasmids expressing VN04-HA-WT or VN04-HA mutants and subjected to ELISA with MAb 9F4 of varying concentrations. Anti-HA5-C PAb was used at a dilution of 1:400 to verify that all the HA proteins were being expressed at similar levels. An irrelevant MAb 1A4 was used at 1.25 mg/ml as a negative control. All absorbance readings were subtracted by the background of H1299 cells transfected with empty vector. Three independent experiments were performed and a representative data set is shown. Data is shown as mean of triplicate wells ±SD. *indicates statistically significant difference of p < 0.05 when compared to VN04-HA-WT. (D) 3D, surface contour representation of the H5 trimer from A/Vietnam/1203/04 (PDB ID: 2FK0). One of the three protomers is emphasized using light e as opposed to dark e grey and is used to display the location of escape residue R62 (blue). Residue F79 (purple), which is spatially close to R62 and has solvent accessibility area, is also shown. Residue W69 (cyan) is difficult to visualize from this perspective, as it is concealed by a more superficial loop. (E) Zoomed in view of (D) in which the protomer is now represented using a ribbon diagram, allowing residue W69 to be more clearly visualized.

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